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Activation of CMA is associated with the relocalization toward the perinuclear region of the lysosomes with higher CMA activity those with higher levels of lys-hsc70 and lamp2a Agarraberes et al. As shown in Figure 4B , in culture fibroblasts maintained in the presence of serum, lamp2a colocalized with other lysosomal membrane proteins lamp1 is shown here , displaying a typical vesicular punctated pattern. When serum was removed from the culture medium, the lamp2a-enriched lysosomes preferentially localized in the perinuclear region.

Treatment with paraquat displayed a similar perinuclear pattern for lamp2a even when the cells were maintained in the presence of serum Figure 4B , right. These results are consistent with a constitutive activation of CMA during oxidative stress in the two experimental models in which CMA has been better characterized, rat liver and fibroblasts in culture Terlecky and Dice, ; Cuervo et al. Although the specific function of each of these cochaperones in CMA remains unknown, studies with human fibroblasts in culture have revealed that the levels of some of them are up-regulated when CMA is activated by removal of serum, whereas others remain unchanged Agarraberes and Dice, In lysosomes isolated from livers of rats starved for 48 h, in addition to the previously reported increase in lys-hsc70, we found higher levels of hsp90, and, in less extent, of Hip, compared with lysosomes from control-fed animals Figure 5A.

Treatment with paraquat in fed rats resulted in similar increase in the lysosomal content of lyshsc70 and of Hip, but only a discrete increase in the content of hsp90, when compared with untreated animals. None of the treatments modified the levels of other associated chaperones hsp40 or Bag-1 , nor did they result in lysosomal association of the inducible form of the hsp70 family, hsp72, which was however elevated in the cytosol after paraquat treatment Figure 5A.

Because the function of hsp90 in lysosomes is not known, it is not clear why the increase in its lysosomal levels is not as pronounced as the one observed during starvation, but because rates of uptake are still up-regulated under these conditions, it points to a nonlimiting role for hsp90 in the translocation complex.

Figure 5. Changes in lysosome-associated chaperones during mild oxidative stress. Levels in lysosomes from fed animals were given an arbitrary value of 1 dotted line. Two components have been shown to be rate-limiting in the uptake of CMA substrates by lysosomes: the chaperone in the lysosomal lumen lys-hsc70 Cuervo et al.

We have previously reported in rat liver the presence of two lysosomal populations with similar morphological and enzymatic characteristics but different CMA activity Cuervo et al. The main difference found, so far, between these two groups of lysosomes is the enrichment of lys-hsc70 in the lumen of the lysosomes with higher CMA activity Cuervo et al. The lysosomes with lower CMA activity can become more active under specific conditions, such as prolonged h starvation or aging, which correlates with an increase in their lumenal levels of lys-hsc70 Cuervo et al.

Because we found higher levels of lyshsc70 in the lysosomes from paraquat-treated animals, we separated these two lysosomal subpopulations to determine whether there was a net increase in lys-hsc70 per lysosome, or it resulted from enrichment of the less active lysosomes in lys-hsc As shown in Figure 5B , after treatment with paraquat there were no significant changes in the content of lys-hsc70 in the membranes or matrices of the less active group.

Accordingly, the ability of this group of lysosomes to selectively take up CMA substrates did not change in paraquat-treated rats our unpublished data. The percentage of hscenriched lysosomes, determined as recovery of hexosaminidase activity in this fraction, was similar in untreated and paraquat-treated rats our unpublished data. For the more active group of lysosomes, as observed during starvation, levels of lys-hsc70 at the lysosomal membrane remained constant, whereas levels of lys-hsc70 in the lysosomal lumen increased approximately fourfold. Separation of the lysosomal membranes and matrices also revealed that hsp90 increased in both compartments during starvation and after paraquat treatment Figure 5B , top , but the increase in the lumen of lysosomes from paraquat-treated rats was smaller than in starved animals.

These differences in the luminal content of hsp90 could explain why, when working with total lysosomes Figure 5A , we found that paraquat induced a lower increase in lysosomal levels of hsp90 than starvation. Whether this hsp90 in the lysosomal lumen is functional, or whether it is only internalized to be degraded amino acid sequence analysis of hsc90 revealed the presence of two KFERQ-related motifs remains unknown. Levels of lamp2a at the lysosomal membrane directly correlate with CMA activity Cuervo and Dice, b.

Lysosomes isolated from mouse fibroblasts treated with H 2 O 2 Figure 6A or from livers of rats exposed to paraquat Figure 6B had higher levels of lamp2a at their membrane than lysosomes isolated from the corresponding untreated controls. This increase seems selective for lamp2a, because levels of other lysosomal membrane proteins lamp1 shown here remained unchanged. In agreement with the binding and uptake data Figure 4A , the increase of lamp2a in the lysosomal membrane induced by paraquat was higher than the one induced by starvation Figure 6C. For the two other lysosomal membrane proteins analyzed, levels of lamp1 remained unchanged in both conditions, and levels of lamp2c were not affected by the treatment with paraquat, but decreased significantly during starvation.

Whether this decrease in lamp2c content is related to the increase in lamp2a or it happens independently is currently under investigation. Figure 6. Lamp2a levels at the lysosomal membrane increase during mild oxidative stress. Memb and lysosomal matrices L. C Imunoblot for lamp2a, lamp2c, and lamp1 of lysosomal membranes isolated from livers of fed rats, h starved Strv rats, or fed rats treated with paraquat PQ. Right, densitometric quantification of six to eight immunoblots as the ones shown here. Levels in lysosomes from fed animals were given an arbitrary value of 1.

We have previously shown that CMA activity can be modulated by changes in the levels of lamp2a at the lysosomal membrane Cuervo and Dice, b. In fact, overexpression of lamp2a results in higher rates of CMA proportional to the increase in the receptor protein Cuervo and Dice, , c. During starvation, the most extensively studied stimuli for CMA, the increase of lamp2a at the lysosomal membrane does not result from de novo synthesis of the protein. Instead, a decrease in the degradation rate of lamp2a, along with the relocation of part of the lamp2a resident in the lysosomal lumen toward the lysosomal membrane account for most of the lamp2a increase in the membrane Cuervo and Dice, , c.

To determine whether similar mechanisms were responsible for the increased levels of lamp2a during oxidative stress, we first compared the rates of degradation of lamp2a in membranes of lysosomes from rats fed, starved, or treated with paraquat. In contrast with the significant reduction in lamp2a degradation induced by starvation, degradation of lamp2a was only slightly slower in rats treated with paraquat than in untreated fed rats Figure 7A.

Likewise, the distribution of lamp2a between membrane and matrix was not significantly affected by the treatment with paraquat, suggesting that the increased levels of lamp2a in the membrane were not the result of recruitment of the lamp2a normally present in the lumen Figure 7B. Figure 7. A novel mechanism for activation of CMA during mild oxidative stress. A Degradation of lamp2a in isolated membranes from normally fed rats, h starved rats, or fed rats treated with paraquat PQ , as indicated under Materials and Methods.

Degradation of lamp2a was followed by immunoblot of the lysosomal membranes with an antibody specific against its cytosolic tail, at different times of the incubation. A representative immunoblot is shown in the top insets. B Distribution of lamp2a between the lysosomal membrane and the matrix in lysosomes isolated from normally fed rats, treated or not with PQ.

C A nucleotide fragment of lamp2a and a nucleotide fragment of actin were amplified, through PCR, from increasing concentrations of total mRNA isolated from the livers of normally fed, h starved, or paraquat-treated fed rats. Values were corrected for actin amplification in each samples and are expressed as fold increase compared to mRNA lamp2a values in fed untreated rats that was given an arbitrary value of 1.

Contrary to starved animals, levels of lamp2a mRNA in the liver of rats treated with paraquat were significantly higher than in untreated rats. Treatment with paraquat induced an increase in lamp2a mRNA levels of approximately sixfold in fed animals, whereas starvation resulted in a slight decrease in lamp2a mRNA levels. Therefore, contrary to starvation, where the increase in lamp2a levels did not require new protein to be synthesized, most of the increase in lamp2a levels detected in paraquat-treated animals was a consequence of de novo synthesis of the protein.

We conclude that activation of CMA is part of the normal oxidative stress response and it contributes to the selective removal of oxidized proteins from the cytosol. The higher rates of CMA observed under these conditions are the combined result of an increase in the susceptibility of the proteins to be taken up and degraded by lysosomes, and an enhanced ability of the lysosomes for substrate uptake.

Unexpectedly, during mild oxidative stress, activation of CMA is mediated by a novel mechanism different from the previously characterized activation of CMA during nutritional stress. Our results provide for the first time evidence for the participation of lysosomes in the removal of oxidized proteins during mild oxidative stress through CMA. This novel role for CMA is supported by the fact that 1 oxidized proteins can be detected in the lumen of lysosomes active for CMA Figure 1 ; 2 in conditions with declined CMA, such as aging, the amount of oxidized proteins translocated into lysosomes is reduced Figure 1B ; 3 oxidized CMA substrates bind and are taken up more efficiently by isolated lysosomes than their unmodified forms Figure 2 ; 4 lysosomes from cells or rats exposed to prooxidants display higher rates of binding and uptake of CMA substrate proteins Figures 3 and 4 ; and 5 blockage of CMA in cultured cells increases their susceptibility to prooxidant compounds and decreases their viability Massey, Kiffin, and Cuervo, unpublished data.

Effects of electromagnetic fields exposure on the antioxidant defense system

Activation of CMA during oxidative-stress is attained through the up-regulation of specific components of the lysosomal translocation complex lysosomal chaperones and the lysosomal membrane receptor Figures 5 and 6. Interestingly, although these changes are similar to the ones described when CMA is activated by nutritional stress, the mechanism involved in the up-regulation is different. Therefore, this finding suggests that stress-mediated activation of CMA varies depending on the nature of the stress.

Most of the previous studies on the lysosomal role during oxidative stress have focused on the contribution of this subcellular compartment to the oxidative damage, rather than on a possible protective role. In our study, we did not find changes in the stability of the lysosomal membrane, assayed either as release of specific enzymes or with a more sensitive proteolytic method. Other possibility is that the initial lysosomal damage still takes place.

In addition, because most of the studies regarding lysosomal stability and oxidative stress have been performed in intact cells, analyzing the leakage of fluorescent probes from the lysosomal compartment, we cannot discard that different lysosomal populations might be differently affected by prooxidants.

Particular characteristics of the CMA-active lysosomes, such as, for example, a lower concentration of iron in their lumen, could explain their higher resistance to prooxidants. Our studies on CMA-active lysosomes in old animals also support their unique characteristics. Thus, although accumulation of lipofuscin in lysosomes is a commonly used biomarker of aging, CMA-active lysosomes from old rat livers rarely accumulate this pigment Cuervo and Dice, a.

The ability of proteasomes to degrade oxidized proteins has been previously well documented in vitro Rivett, ; Davies, , and more recently in vivo Grune et al. Most of the studies assessing oxidation-induced changes in lysosomal proteolytic behavior have focused in the analysis of the enzymatic activity of cathepsins, the lysosomal proteases. Moderate oxidative stress does not significantly change the activity of most cathepsins, whereas more severe oxidizing conditions result in increased or decreased cathepsin activity depending on the cellular conditions Sitte et al.

This lack of correlation between the intracellular accumulation of oxidized proteins and the activity of lysosomal enzymes sets the basis for arguments against the lysosomal participation in removal of oxidized proteins Sitte et al. However, the activity of the lysosomal proteases is a nonlimiting step for any of the forms of autophagy. The intralysosomal concentration of cathepsins is such that, once substrates reach the lysosomal lumen, they are rapidly degraded. This makes the delivery of substrates the limiting step.

Bergamini and colleagues were one of the first groups to point out that, if the degradation of proteins by lysosomes, instead of the enzymatic activity of lysosomal proteases, is considered, there is a good inverse correlation between lysosomal proteolysis and intracellular content of oxidized proteins Vittorini et al. We have previously shown that, in the group of lysosomes active for CMA, the activity of most lysosomal enzymes does not change significantly with age, and yet rates of CMA are lower in aged cells Cuervo and Dice, a.

The impaired ability of this group of lysosomes to take up substrates in older animals could explain why there is a lower content of oxidized proteins in their lumen, despite the higher level of oxidized cytosolic proteins Figure 1B. It is unlikely that the lower levels of oxidized proteins detected in the lysosomal lumen of old rats, result from faster degradation of these proteins inside lysosomes, because even when we inhibited lysosomal degradation in rats, before lysosomal isolation by i. The contribution of proteasomes to the removal of oxidized proteins in vivo has been demonstrated by analyzing the consequences of blocking its catalytic activity in oxidized protein removal Grune et al.

This approach, however, cannot be used to evaluate the contribution of autophagy in this process. Even if the lysosomal proteases are inhibited either with specific protease inhibitors or by raising the intralysosomal pH , we do not expect to find changes in the cytosolic content of oxidized proteins, because only their proteolysis, but not their translocation into the lysosomal lumen, would be blocked. The accumulation of undegraded products in the lysosomal lumen can be tolerated for a long time without affecting their translocation ability.

An alternative approach would be to directly inhibit the delivery of the substrates. No chemical inhibitors are available to block CMA. We have recently succeeded in blocking substrate translocation by using RNA interference against the lysosomal receptor in cultured fibroblasts Massey, Kiffin, and Cuervo, unpublished data. As mentioned before, these cells have a lower resistance to oxidative stress. Likewise, chronic inhibition of proteasomes, similar to the one that occurs in aging, alters the ability of the cells to activate autophagy in response to stress Ding et al.


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Although an experimental challenge, this cross-talking among the different proteolytic systems offers perhaps a more physiological view of what is really happening in the cells during oxidative stress. In the same way that the relative contribution of proteasomes and lysosomes to total protein degradation varies depending on the cell type and on the cellular conditions Fuertes et al. In fact, there are now numerous examples of proteins that can be degraded by more than one proteolytic pathway Cuervo et al.

Future studies should be directed at understanding how those fluctuations are regulated and to identify possible ways of stimulating one system to compensate for the failure of another. Whereas during severe oxidative stress, protein aggregation and cross-linking are common events, mild oxidative stress has been shown to generate excellent proteolytic substrates Grune et al. Although the exact mechanism behind this facilitated uptake is still elusive, we hypothesize that protein unfolding, likely to occur during oxidation Imai et al.

Oxidized substrates were taken up faster by lysosomes even when we did not add cytosolic hsc70 in the translocation cocktail our unpublished data. Our unpublished data showing that manipulations, such as partial denaturation of CMA substrates or extensive truncation, increase their rates of uptake into lysosomes via CMA Salvador, Aguado, Cuervo, and Knecht, unpublished data , also support the proposed facilitating role of partial protein unfolding under mild oxidative conditions.

Whether the activation of CMA under these conditions is a response to the presence of these partially unfolded proteins in the cytosol, or it is mediated by other cytosolic components generated during the oxidative stress remains to be elucidated. An interesting remaining question is how the cells decide whether an oxidized protein should be degraded by the proteasome or in the lysosomal compartment. However, a very intriguing idea proposed by Gracy et al. As other targeting motifs, the KFERQ-like motif is degenerate, resulting from the combination of a basic, a hydrophobic and an acid residue with a fourth basic or hydrophobic residue, flanked on either side by a glutamine.

Oxidation for example of a histidine to aspartic acid could provide the acid residue necessary to complete a KFERQ-like motif. On the other hand, the same process could eliminate KFERQ-like motifs in proteins that normally are substrates for this pathway, resulting in their impaired degradation in conditions such as aging. This modification of the targeting motif, added to the fact that CMA activity decreases with age, could explain the lower content of oxidized proteins in the lumen of CMA active lysosomes in old rats, despite the higher levels of oxidized proteins in their cytosol.

Our laboratory is currently trying to identify changes in the targeting motif of CMA substrates with age using a shot-gun proteomic approach. Particularly exciting is the finding that although activation of CMA during oxidative and nutritional stress have the same consequences on the levels of the lysosomal translocation components, up-regulation, at least for the lysosomal receptor, obeys different mechanisms. During starvation CMA is activated to supply the cells with amino acids required for the synthesis of essential proteins.

Under these conditions, de novo synthesis of lamp2a to increase CMA rates is probably not a reasonable option due to the shortage of amino acids. Instead, more conservative mechanisms down-regulation of lamp2a degradation and intralysosomal relocation are adopted Cuervo and Dice, b. If oxidative damage takes place under normal nutritional conditions, as the supply of amino acids would not be compromised, de novo synthesis of lamp2a is up-regulated to activate CMA Figure 7C.

De novo synthesis might be advantageous under these conditions, because it provides a faster mechanism for CMA activation. In conclusion, we have identified for the first time a role of CMA as part of the oxidative stress response. Because CMA activity is severely impaired during aging, we hypothesize that part of the accumulation of oxidized proteins observed in old organisms may result from the malfunctioning of CMA.

Future efforts aimed to restore normal CMA activity in old cells would help to understand the relevance of the cross-talk among different proteolytic pathways, and the compensatory mechanisms activated when the activity of one or more of these pathways is compromised. Article published online ahead of print.

Cell Article and publication date are available at www. Monitoring Editor: Jennifer Lippincott-Schwartz. We gratefully acknowledge Dr. Fernando Macian and the other members of our laboratory for valuable suggestions and comments. Molecular Biology of the Cell Vol. This is the final version - click for previous version. Add to favorites Download Citations Track Citations. Abstract Oxidatively damaged proteins accumulate with age in almost all cell types and tissues. A molecular chaperone complex at the lysosomal membrane is required for protein translocation.

Cell Sci. An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation.

1. Introduction

Cell Biol. Uptake and degradation of glyceraldehydephosphate dehydrogenase by rat liver lysosomes. Therefore, contrary to starvation, where the increase in lamp2a levels did not require new protein to be synthesized, most of the increase in lamp2a levels detected in paraquat-treated animals was a consequence of de novo synthesis of the protein. We conclude that activation of CMA is part of the normal oxidative stress response and it contributes to the selective removal of oxidized proteins from the cytosol.

The higher rates of CMA observed under these conditions are the combined result of an increase in the susceptibility of the proteins to be taken up and degraded by lysosomes, and an enhanced ability of the lysosomes for substrate uptake. Unexpectedly, during mild oxidative stress, activation of CMA is mediated by a novel mechanism different from the previously characterized activation of CMA during nutritional stress.

Our results provide for the first time evidence for the participation of lysosomes in the removal of oxidized proteins during mild oxidative stress through CMA. This novel role for CMA is supported by the fact that 1 oxidized proteins can be detected in the lumen of lysosomes active for CMA Figure 1 ; 2 in conditions with declined CMA, such as aging, the amount of oxidized proteins translocated into lysosomes is reduced Figure 1B ; 3 oxidized CMA substrates bind and are taken up more efficiently by isolated lysosomes than their unmodified forms Figure 2 ; 4 lysosomes from cells or rats exposed to prooxidants display higher rates of binding and uptake of CMA substrate proteins Figures 3 and 4 ; and 5 blockage of CMA in cultured cells increases their susceptibility to prooxidant compounds and decreases their viability Massey, Kiffin, and Cuervo, unpublished data.

Activation of CMA during oxidative-stress is attained through the up-regulation of specific components of the lysosomal translocation complex lysosomal chaperones and the lysosomal membrane receptor Figures 5 and 6. Interestingly, although these changes are similar to the ones described when CMA is activated by nutritional stress, the mechanism involved in the up-regulation is different.

Therefore, this finding suggests that stress-mediated activation of CMA varies depending on the nature of the stress. Most of the previous studies on the lysosomal role during oxidative stress have focused on the contribution of this subcellular compartment to the oxidative damage, rather than on a possible protective role.

In our study, we did not find changes in the stability of the lysosomal membrane, assayed either as release of specific enzymes or with a more sensitive proteolytic method. Other possibility is that the initial lysosomal damage still takes place. In addition, because most of the studies regarding lysosomal stability and oxidative stress have been performed in intact cells, analyzing the leakage of fluorescent probes from the lysosomal compartment, we cannot discard that different lysosomal populations might be differently affected by prooxidants.

Particular characteristics of the CMA-active lysosomes, such as, for example, a lower concentration of iron in their lumen, could explain their higher resistance to prooxidants. Our studies on CMA-active lysosomes in old animals also support their unique characteristics. Thus, although accumulation of lipofuscin in lysosomes is a commonly used biomarker of aging, CMA-active lysosomes from old rat livers rarely accumulate this pigment Cuervo and Dice, a.

The ability of proteasomes to degrade oxidized proteins has been previously well documented in vitro Rivett, ; Davies, , and more recently in vivo Grune et al. Most of the studies assessing oxidation-induced changes in lysosomal proteolytic behavior have focused in the analysis of the enzymatic activity of cathepsins, the lysosomal proteases. Moderate oxidative stress does not significantly change the activity of most cathepsins, whereas more severe oxidizing conditions result in increased or decreased cathepsin activity depending on the cellular conditions Sitte et al.

This lack of correlation between the intracellular accumulation of oxidized proteins and the activity of lysosomal enzymes sets the basis for arguments against the lysosomal participation in removal of oxidized proteins Sitte et al. However, the activity of the lysosomal proteases is a nonlimiting step for any of the forms of autophagy. The intralysosomal concentration of cathepsins is such that, once substrates reach the lysosomal lumen, they are rapidly degraded. This makes the delivery of substrates the limiting step.

Bergamini and colleagues were one of the first groups to point out that, if the degradation of proteins by lysosomes, instead of the enzymatic activity of lysosomal proteases, is considered, there is a good inverse correlation between lysosomal proteolysis and intracellular content of oxidized proteins Vittorini et al.

We have previously shown that, in the group of lysosomes active for CMA, the activity of most lysosomal enzymes does not change significantly with age, and yet rates of CMA are lower in aged cells Cuervo and Dice, a. The impaired ability of this group of lysosomes to take up substrates in older animals could explain why there is a lower content of oxidized proteins in their lumen, despite the higher level of oxidized cytosolic proteins Figure 1B.

It is unlikely that the lower levels of oxidized proteins detected in the lysosomal lumen of old rats, result from faster degradation of these proteins inside lysosomes, because even when we inhibited lysosomal degradation in rats, before lysosomal isolation by i. The contribution of proteasomes to the removal of oxidized proteins in vivo has been demonstrated by analyzing the consequences of blocking its catalytic activity in oxidized protein removal Grune et al.

This approach, however, cannot be used to evaluate the contribution of autophagy in this process. Even if the lysosomal proteases are inhibited either with specific protease inhibitors or by raising the intralysosomal pH , we do not expect to find changes in the cytosolic content of oxidized proteins, because only their proteolysis, but not their translocation into the lysosomal lumen, would be blocked. The accumulation of undegraded products in the lysosomal lumen can be tolerated for a long time without affecting their translocation ability.

An alternative approach would be to directly inhibit the delivery of the substrates. No chemical inhibitors are available to block CMA. We have recently succeeded in blocking substrate translocation by using RNA interference against the lysosomal receptor in cultured fibroblasts Massey, Kiffin, and Cuervo, unpublished data. As mentioned before, these cells have a lower resistance to oxidative stress. Likewise, chronic inhibition of proteasomes, similar to the one that occurs in aging, alters the ability of the cells to activate autophagy in response to stress Ding et al.

Although an experimental challenge, this cross-talking among the different proteolytic systems offers perhaps a more physiological view of what is really happening in the cells during oxidative stress. In the same way that the relative contribution of proteasomes and lysosomes to total protein degradation varies depending on the cell type and on the cellular conditions Fuertes et al.

In fact, there are now numerous examples of proteins that can be degraded by more than one proteolytic pathway Cuervo et al. Future studies should be directed at understanding how those fluctuations are regulated and to identify possible ways of stimulating one system to compensate for the failure of another. Whereas during severe oxidative stress, protein aggregation and cross-linking are common events, mild oxidative stress has been shown to generate excellent proteolytic substrates Grune et al.

Although the exact mechanism behind this facilitated uptake is still elusive, we hypothesize that protein unfolding, likely to occur during oxidation Imai et al. Oxidized substrates were taken up faster by lysosomes even when we did not add cytosolic hsc70 in the translocation cocktail our unpublished data.

Our unpublished data showing that manipulations, such as partial denaturation of CMA substrates or extensive truncation, increase their rates of uptake into lysosomes via CMA Salvador, Aguado, Cuervo, and Knecht, unpublished data , also support the proposed facilitating role of partial protein unfolding under mild oxidative conditions. Whether the activation of CMA under these conditions is a response to the presence of these partially unfolded proteins in the cytosol, or it is mediated by other cytosolic components generated during the oxidative stress remains to be elucidated.

An interesting remaining question is how the cells decide whether an oxidized protein should be degraded by the proteasome or in the lysosomal compartment. However, a very intriguing idea proposed by Gracy et al. As other targeting motifs, the KFERQ-like motif is degenerate, resulting from the combination of a basic, a hydrophobic and an acid residue with a fourth basic or hydrophobic residue, flanked on either side by a glutamine.

Oxidation for example of a histidine to aspartic acid could provide the acid residue necessary to complete a KFERQ-like motif. On the other hand, the same process could eliminate KFERQ-like motifs in proteins that normally are substrates for this pathway, resulting in their impaired degradation in conditions such as aging. This modification of the targeting motif, added to the fact that CMA activity decreases with age, could explain the lower content of oxidized proteins in the lumen of CMA active lysosomes in old rats, despite the higher levels of oxidized proteins in their cytosol.

Our laboratory is currently trying to identify changes in the targeting motif of CMA substrates with age using a shot-gun proteomic approach. Particularly exciting is the finding that although activation of CMA during oxidative and nutritional stress have the same consequences on the levels of the lysosomal translocation components, up-regulation, at least for the lysosomal receptor, obeys different mechanisms.

During starvation CMA is activated to supply the cells with amino acids required for the synthesis of essential proteins. Under these conditions, de novo synthesis of lamp2a to increase CMA rates is probably not a reasonable option due to the shortage of amino acids. Instead, more conservative mechanisms down-regulation of lamp2a degradation and intralysosomal relocation are adopted Cuervo and Dice, b. If oxidative damage takes place under normal nutritional conditions, as the supply of amino acids would not be compromised, de novo synthesis of lamp2a is up-regulated to activate CMA Figure 7C.

De novo synthesis might be advantageous under these conditions, because it provides a faster mechanism for CMA activation. In conclusion, we have identified for the first time a role of CMA as part of the oxidative stress response. Because CMA activity is severely impaired during aging, we hypothesize that part of the accumulation of oxidized proteins observed in old organisms may result from the malfunctioning of CMA.

Future efforts aimed to restore normal CMA activity in old cells would help to understand the relevance of the cross-talk among different proteolytic pathways, and the compensatory mechanisms activated when the activity of one or more of these pathways is compromised. Article published online ahead of print.

Oxidative stress - Wikipedia

Cell Article and publication date are available at www. Monitoring Editor: Jennifer Lippincott-Schwartz. We gratefully acknowledge Dr. Fernando Macian and the other members of our laboratory for valuable suggestions and comments. Molecular Biology of the Cell Vol. This is the final version - click for previous version. Add to favorites Download Citations Track Citations.

Abstract Oxidatively damaged proteins accumulate with age in almost all cell types and tissues. A molecular chaperone complex at the lysosomal membrane is required for protein translocation. Cell Sci. An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation. Cell Biol. Uptake and degradation of glyceraldehydephosphate dehydrogenase by rat liver lysosomes. Exposure of cells to nonlethal concentrations of hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal destabilization.

Free Radic. Hypochlorous acid-modified low-density lipoprotein inactivates the lysosomal protease cathepsin B: protection by ascorbic and lipoic acids. Redox Rep. Impairment of proteasome structure and function in aging. Peptide sequences that target proteins for enhanced degradation during serum withdrawal. Google Scholar Chiang, H. A role for a kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science , Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues. Protein Sci. Autophagy: in sickness and in health.

Trends Cell Biol. Autophagy: many paths to the same end. A receptor for the selective uptake and degradation of proteins by lysosomes. How do intracellular proteolytic systems change with age? Google Scholar Cuervo, A. Lysosomes, a meeting point of proteins, chaperones, and proteases. Age-related decline in chaperone-mediated autophagy. Regulation of lamp2a levels in the lysosomal membrane. Traffic 1 , Unique properties of lamp2a compared to other lamp2 isoforms.

A population of rat liver lysosomes responsible for the selective uptake and degradation of cytosolic proteins. Direct lysosomal uptake of alpha2-microglobulin contributes to chemically induced nephropathy. Kidney Int. IkB is a substrate for a selective pathway of lysosomal proteolysis. Cell 9 , Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation. Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor.

EMBO J. Selective binding and uptake of ribonuclease A and glyceraldehydephosphate dehydrogenase by rat liver lysosomes. Degradation of oxidized proteins by the 20S proteasome. Biochimie 83 , Proteasome inhibitors induce intracellular protein aggregation and cell death by an oxygen-dependent mechanism. FEBS Lett. Chaperone-mediated autophagy. In: Autophagy , ed. Google Scholar Dice, J. Google Scholar Ding, Q. Characterization of chronic low-level proteasome inhibition on neural homeostasis. Age-related changes in the autophagic proteolysis of rat isolated liver cells: effects of antiaging dietary restrictions.

Google Scholar Dunlop, R. Recent developments in the intracellular degradation of oxidized proteins. A mechanism regulating proteolysis of specific proteins during renal tubular cell growth. Protein repair and degradation during aging. World J. Google Scholar Friguet, B. Protein degradation by the proteasome and its implications in aging. Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions.

Induction of protein catabolism and the ubiquitin-proteasome pathway by mild oxidative stress. Cancer Lett. Molecular wear and tear leads to terminal marking and the unstable isoforms of aging. Oxidative stress, aging and the proteasomal system. Biogerontology 1 , Levels of 8-iso-PGF2-alpha are likely to be useful in predicting oxidative status in diseases such as endometriosis, and might be instrumental in determining the cause of concurrent infertility. Both forms have been reported as individual markers of different pathological processes [ 92 ].

Heat shock protein 70 B is an inducible member of HSP family that is present in low levels under normal conditions [ 93 ] and in high levels [ 94 ] under situations of stress. It functions as a chaperone for proteostatic processes such as folding and translocation, while maintaining quality control [ 95 ].


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  • It has also been noted to promote cell proliferation through the suppression of apoptosis, especially when expressed in high levels, as noted in many tumor cells [ 94 , 96 - 98 ]. As such, HSP70 is overexpressed when there is an increased number of misfolded proteins, and thus, an overabundance of ROS [ 94 ]. TLR 4 , possibly accounting for pelvic inflammation and growth of endometriotic tissue [ 99 ]. Fragmentation of HSP70 has been suggested to result in unregulated expression of transcription factor NF-kappa B [ ], which may further promote inflammation within the pelvic cavity of patients with endometriosis.

    Oxidants have been proposed to encourage growth of ectopic endometrial tissue through the induction of cytokines and growth factors [ ]. Signaling mediated by NF-kappa B stimulates inflammation, invasion, angiogenesis, and cell proliferation; it also prevents apoptosis of endometriotic cells. Activation of NF-kappa B by OS has been detected in endometriotic lesions and peritoneal macrophages of patients with endometriosis [ ].

    N-acetylcysteine NAC and vitamin E are antioxidants that limit the proliferation of endometriotic cells [ ], likely by inhibiting activation of NF-kappa B [ ]. Future studies may implicate a therapeutic effect of NAC and vitamin E supplementation on endometriotic growth. This may explain the increased expressions of these proteins in ectopic versus eutopic endometrial tissue [ ].

    In the peritoneum of patients with endometriosis, accumulation of iron and heme around endometriotic lesions [ ] from retrograde menstruation [ ] up-regulates iNOS activity and generation of NO by peritoneal macrophages [ ]. Extensive degradation of DNA by iron and heme accounts for their considerable free radical activity. Chronic oxidative insults from iron buildup within endometriotic lesions may be a key factor in the development of the disease [ ].

    Naturally, endometriotic cysts contain high levels of free iron as a result of recurrent cyclical hemorrhage into them compared to other types of ovarian cysts. However, high concentrations of lipid peroxides, 8-OHdG, and antioxidant markers in endometrial cysts indicate lipid peroxidation, DNA damage, and up-regulated antioxidant defenses respectively.

    These findings strongly suggest altered redox status within endometrial cysts [ ]. Based on results from their studies of human endometrium, Kobayashi et al have proposed a role for iron chelators such as dexrazoxane, deferoxamine, and deferasirox to prevent the accumulation of iron in and around endometriotic lesions [ ]. Future studies investigating the use of iron chelators may prove beneficial in the prevention of lesion formation and the reduction of lesion size. Many genes encoding antioxidant enzymes and proteins are recruited to combat excessive ROS and to prevent cell damage.

    Amongst these are Trx and Trx reductase, which sense altered redox status and help maintain cell survival against ROS [ ]. Total thiol levels, used to predict total antioxidant capacity TAC , have been found to be decreased in women with pelvic endometriosis and may contribute to their status of OS [ 81 , ].

    Conversely, results from a more recent study failed to correlate antioxidant nutrients with total thiol levels [ ]. Patients with endometriosis tend to have lower pregnancy rates than women without the disease. Low oocyte and embryo quality in addition to spermatotoxic peritoneal fluid may be mediated by ROS and contribute to the subfertility experienced by patients with endometriosis [ ].

    The peritoneal fluid of women with endometriosis contains low concentrations of the antioxidants ascorbic acid [ 82 ] and GPx [ 81 ]. The reduction in GPx levels was proposed to be secondary to decreased progesterone response of endometrial cells [ ]. The link between gene expression for progesterone resistance and OS may facilitate a better understanding of the pathogenesis of endometriosis.

    It has been suggested that diets lacking adequate amounts of antioxidants may predispose some women to endometriosis [ ]. Studies have shown decreased levels of OS markers in people who consume antioxidant rich diets or take antioxidant supplements [ - ]. In certain populations, women with endometriosis have been observed to have a lower intake of vitamins A, C [ ], E [ - ], Cu, and Zn [ ] than fertile women without the disease [ - ]. Daily supplementation with vitamins C and E for 4 months was found to decrease levels of OS markers in these patients, and was attributed to the increased intake of these vitamins and their possible synergistic effects.

    Pregnancy rates, however, did not improve [ ]. Intraperitoneal administration of melatonin, a potent scavenger of free radicals, has been shown to cause regression of endometriotic lesions [ - ] by reducing OS [ , ]. These findings, however, were observed in rodent models of endometriosis, which may not closely resemble the disease in humans. It is evident that endometriotic cells contain high levels of ROS; however, their precise origins remain unclear.

    Impaired detoxification processes lead to excess ROS and OS, and may be involved in increased cellular proliferation and inhibition of apoptosis in endometriotic cells. It is a disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovaries [ ]. Clinical manifestations of PCOS commonly include menstrual disorders, which range from amenorrhea to menorrhagia. Skin disorders are also very prevalent amongst these women.

    Insulin resistance may be central to the etiology of PCOS. Signs of insulin resistance such as hypertension, obesity, and central fat distribution are associated with other serious conditions, such as metabolic syndrome, nonalcoholic fatty liver [ ], and sleep apnea. All of these conditions are risk factors for long-term metabolic sequelae, such as cardiovascular disease and diabetes [ ].

    Most importantly, waist circumference, independent of body mass index BMI , is responsible for an increase in oxLDL [ 71 ]. Polycystic ovary syndrome is also associated with decreased antioxidant concentrations, and is thus considered an oxidative state [ ]. The mononuclear cells of women with PCOS are increased in this inflammatory state [ ], which occurs more so from a heightened response to hyperglycemia and C-reactive protein CRP. Physiological hyperglycemia generates increased levels of ROS from mononuclear cells, which then activate the release of TNF-alpha and increase inflammatory transcription factor NF-kappa B.

    As a result, concentrations of TNF-alpha, a known mediator of insulin resistance, are further increased. The resultant OS creates an inflammatory environment that further increases insulin resistance and contributes to hyperandrogenism [ ]. Lifestyle modification is the cornerstone treatment for women with PCOS. This includes exercise and a balanced diet, with a focus on caloric restriction [ ].

    However, if lifestyle modifications do not suffice, a variety of options for medical therapy exist. Combined oral contraceptives are considered the primary treatment for menstrual disorders. Currently, there is no clear primary treatment for hirsutism, although it is known that combination therapies seem to produce better results [ ]. Unexplained infertility is defined as the inability to conceive after 12 months of unprotected intercourse in couples where known causes of infertility have been ruled out.

    It is thus considered a diagnosis of exclusion. Its pathophysiology remains unclear, although the literature suggests a possible contribution by increased levels of ROS, especially shown by increased levels of the lipid peroxidation marker, MDA [ , ] in comparison to antioxidant concentration in the peritoneal cavity [ ].

    The increased amounts of ROS in these patients are suggestive of a reduction in antioxidant defenses, including GSH and vitamin E [ 76 ]. The low antioxidant status of the peritoneal fluid may be a determinant factor in the pathogenesis of idiopathic infertility. N-acetyl cysteine is a powerful antioxidant with anti-apoptotic effects.

    It is known to preserve vascular integrity and to lower levels of homocysteine, an inducer of OS and apoptosis. Badaiwy et al conducted a randomized, controlled, study in which NAC was compared with clomiphene citrate as a cofactor for ovulation induction in women with unexplained infertility [ ]. The study, however, concluded that NAC was ineffective in inducing ovulation in patients in these patients [ ]. Folate is a B9 vitamin that is considered indispensable for reproduction. It plays a role in amino acid metabolism and the methylation of proteins, lipids, and nucleic acids.

    Acquired or hereditary folate deficiency contributes to homocysteine accumulation. Polymorphisms in folate-metabolizing pathways of genes may account for the unexplained infertility seen in these women, as it disrupts homocysteine levels and subsequently alters homeostatic status. Impaired folate metabolism disturbs endometrial maturation and results in poor oocyte quality [ ]. More studies are clearly needed to explore the efficacy of antioxidant supplementation as a possible management approach for these patients.

    The placenta is a vital organ of pregnancy that serves as a maternal-fetal connection through which nutrient, O 2 , and hormone exchanges occur. It also provides protection and immunity to the developing fetus. In humans, normal placentation begins with proper trophoblastic invasion of the maternal spiral arteries and is the key event that triggers the onset of these placental activities [ 6 ]. The placental vasculature undergoes changes to ensure optimal maternal vascular perfusion.

    Prior to the unplugging of the maternal spiral arteries by trophoblastic plugs, the state of low O 2 tension in early pregnancy gives rise to normal, physiological hypoxia [ ]. During this time, the syncytiotrophoblast is devoid of antioxidants, and thus, remains vulnerable to oxidative damage [ , ]. Between 10 and 12 weeks of gestation, the trophoblastic plugs are dislodged from the maternal spiral arteries, flooding the intervillous space with maternal blood.

    This event is accompanied by a sharp rise in O 2 tension [ ], marking the establishment of full maternal arterial circulation to the placenta associated with an increase in ROS, which leads to OS [ 68 ]. At physiological concentrations, ROS stimulate cell proliferation and gene expression [ ]. Placental acclimation to increased O 2 tension and OS at the end of the 1 st trimester up-regulates antioxidant gene expression and activity to protect fetal tissue against the deleterious effects of ROS during the critical phases of embryogenesis and organogenesis [ 2 ].

    If maternal blood flow reaches the intervillous space prematurely, placental OS can ensue too early and cause deterioration of the syncytiotrophoblast. This may give rise to a variety of complications including miscarriage [ , , ], recurrent pregnancy loss [ ], and preeclampsia, amongst others [ ]. These complications will be discussed below. Congenital anomalies and maternal factors such as uterine anomalies, infection, diseases, and idiopathic causes constitute the remaining causes [ ]. Overwhelming placental OS has been proposed as a causative factor of spontaneous abortion.

    As mentioned earlier, placentas of normal pregnancies experience an oxidative burst between 10 and 12 weeks of gestation. This OS returns to baseline upon the surge of antioxidant activity, as placental cells gradually acclimate to the newly oxidative surroundings [ ]. In cases of miscarriage, the onset of maternal intraplacental circulation occurs prematurely and sporadically between 8 and 9 weeks of pregnancy in comparison to normal continuous pregnancies [ , ]. In these placentas, high levels of HSP70, nitrotyrosine [ , ], and markers of apoptosis have been reported in the villi, suggesting oxidative damage to the trophoblast with subsequent termination of the pregnancy [ 2 ].

    Antioxidant enzymes are unable to counter increases in ROS at this point, since their expression and activity increases with gestational age [ ]. The activity of serum prolidase, a biomarker of extracellular matrix and collagen turnover, has been observed to be decreased in patients with early pregnancy loss. Its levels were also shown to negatively correlate with increased OS, possibly accounting for the heightened placental vascular resistance and endothelial dysfunction secondary to decreased and dysregulated collagen turnover [ ].

    A negative correlation with lipid hydroperoxide was also observed in these patients, indicating their high susceptibility to lipid peroxidation [ ]. Oxidative stress can also affect homeostasis in the ER. Persistence of endoplasmic OS can further sustain ER stress, eventually increasing decidual cell apoptosis and resulting in early pregnancy loss [ ]. Decreased detoxification ability of GPx may occur in the setting of Se deficiency, which has been linked to both spontaneous abortion [ , ] and recurrent pregnancy loss [ ].

    Apoptosis of placental tissues may result from OS-induced inflammatory processes triggered by a variety of factors. Several etiologies may underlie improper initiation of maternal blood flow to the intervillous space; yet it may be through this mechanism by which both spontaneous and recurrent pregnancy loss occur. Antioxidant supplementation has been investigated in the prevention of early pregnancy loss, with the idea of replacing depleted antioxidant stores to combat an overwhelmingly oxidative environment.

    However, a meta-analysis of relevant studies failed to report supporting evidence of beneficial effects of antioxidant supplementation [ ]. It has been more recently suggested that the maternal uterine spiral arteries of normal pregnancies may involve uterine natural killer NK cells as a regulator of proper development and remodeling. Angiogenic factors are known to play key roles in the maintenance of proper spiral artery remodeling. Thus, the involvement of uterine NK cells in RPL has been supported by the early pregnancy findings of increased levels of angiogenic factors secreted by uterine NK cells [ ], as well as increased in vivo and in vitro endothelial cell angiogenesis induced by uterine NK cells [ ] in patients with RPL.

    Women experiencing RPL have also been noted to have increased endometrial NK cells, which were positively correlated to endometrial vessel density. Accordingly, it has been suggested that an increase of uterine NK cells increases pre-implantation angiogenesis, leading to precocious intra-placental maternal circulation, and consequently, significantly increased OS early in pregnancy [ ].

    The syncytiotrophoblastic deterioration and OS that occur as a result of abnormal placentation may explain the heightened sensitivity of syncytiotrophoblasts to OS during the 1 st trimester, and could contribute significantly to idiopathic RPL [ ]. Furthermore, markedly increased levels of GSH have also been found in the plasma of women with a history of RPL, indicating a response to augmented OS [ ]. Polymorphisms of antioxidant enzymes have been associated with a higher risk of RPL [ - ]. Antioxidant supplementation may be the answer to restoring antioxidant defenses and combating the effects of placental apoptosis and inflammatory responses associated with extensive OS.

    In addition to its well-known antioxidant properties, NAC is rich in sulphydryl groups. Its thiol properties give it the ability to increase intracellular concentrations of GSH or directly scavenge free radicals [ , ]. Furthermore, the fetal toxicity, death in utero, and IUGR, induced by lipopolysaccharides, might be prevented by the antioxidant properties of NAC [ ]. By inhibiting the release of pro-inflammatory cytokines [ ], endothelial apoptosis, and oxidative genotoxicity [ ], via maintenance of intracellular GSH levels, NAC may well prove promising to suppress OS-induced reactions and processes responsible for the oxidative damage seen in complicated pregnancies.

    Preeclampsia is a complex multisystem disorder that can affect previously normotensive women. Preeclampsia can develop before early onset or after late onset 34 weeks of gestation. The major pathophysiologic disturbances are focal vasospasm and a porous vascular tree that transfers fluid from the intravascular to the extravascular space. The exact mechanism of vasospasm is unclear, but research has shown that interactions between vasodilators and vasoconstrictors, such as NO, endothelin 1, angiotensin II, prostacyclin, and thromboxane, can cause decrease the perfusion of certain organs.

    The porous vascular tree is one of decreased colloid osmotic pressure and increased vascular permeability [ - ]. From early pregnancy on, the body assumes a state of OS. Oxidative stress is important for normal physiological functions and for placental development [ ]. Preeclampsia, however, represents a much higher state of OS than normal pregnancies do [ ].

    Early-onset preeclampsia is associated with elevated levels of protein carbonyls, lipid peroxides, nitrotyrosine residues, and DNA oxidation, which are all indicators of placental OS [ 68 , ]. The OS of preeclampsia is thought to originate from insufficient spiral artery conversion [ , , ] which leads to discontinuous placental perfusion and a low-level ischemia-reperfusion injury [ , , ].

    Ischemia-reperfusion injury stimulates trophoblastic and endothelial cell production of ROS [ ], along with variations in gene expression that are similar to those seen in preeclampsia [ 3 ]. Oxidative stress can cause increased nitration of p38 MAPK, resulting in a reduction of its catalytic activity. This may cause the poor implantation and growth restriction observed in preeclampsia [ 6 ]. Exaggerated apoptosis of villous trophoblasts has been identified in patients with preeclampsia, of which OS has been suggested as a possible contributor.

    Microparticles of syncytiotrophoblast microvillus membrane STBMs have been found throughout the maternal circulation of patients with preeclampsia and are known to cause endothelial cell injury in vitro [ ]. Increased circulating levels of the vasoconstrictor H 2 O 2 [ , ] and decreased levels of the vasodilator NO [ , ] have been noted in preeclampsia and may account for the vasoconstriction and hypertension present in the disease. Still, some studies have conversely reported increased circulating [ , ] and placental [ ] NO levels. Neutrophil modulation occurring in preeclampsia is another important source of ROS, and results in increased production of the SO anion and decreased NO release, which ultimately cause endothelial cell damage in patients with preeclampsia [ ].

    Elevated circulating levels of sFlt-1 have been suggested to play a role in the pathogenesis of preeclampsia [ , ] and the associated endothelial dysfunction [ ]. Placental trophoblastic hypoxia resulting in OS has been linked to excess sFlt-1 levels in the circulation of preeclamptic women [ ]. Vitamins C and E, and sulfasalazine can decrease sFlt-1 levels [ ].

    Heme oxygenase-1 [ ] is an antioxidant enzyme that has anti-inflammatory and cytoprotective properties. Hypoxia stimulates the expression of HO-1 [ ] in cultured trophoblastic cells, and is used to detect increased OS therein [ ]. Preeclampsia may be associated with decreased levels of HO in the placenta [ ], suggesting a decline in protective mechanisms in the disease. Tissue from chorionic villous sampling of pregnant women who were diagnosed with preeclampsia later in gestation revealed considerably decreased expressions of HO-1 and SOD [ ].

    Failure to neutralize overwhelming OS may result in diminished antioxidant defenses. Members of the family of NAD P H oxidases are important generators of the SO anion in many cells, including trophoblasts and vascular endothelial cells. Increased SO anion production through activation of these enzymes may occur through one of several physiological mechanisms, and has been implicated in the pathogenesis of some vascular diseases [ ].

    In cultured trophoblast and smooth muscle cells, the AT1 receptor of preeclamptic women has been observed to promote both the generation of the SO anion and overexpression of NAD P H oxidase [ ]. Between 6 and 8 weeks of gestation, active placental NAD P H yields significantly more SO anion than is produced during full-term [ ]. Thus, early placental development may be affected through dysregulated vascular development and function secondary to NAD P H oxidase-mediated altered gene expression [ 48 , ].

    More specifically, it has been reported that women with early-onset preeclampsia produce higher amounts of the SO anion than women with late-onset disease [ ]. The mechanism of placental NAD P H activation is still unclear, but the above findings may assist in elucidating the role of OS in the pathogenesis of placental dysfunction in reproductive diseases such as preeclampsia.

    Baker et al demonstrated that PON 1 levels tend to be high in patients with preeclampsia, which suggests that OS contributes to the pathogenesis of the disease [ ]. Paraoxonase-1 has also been measured to be increased in patients in mid-gestation [ ], possibly in an attempt to shield against the toxic effects of high OS encountered in preeclampsia. In contrast, other studies have observed considerably decreased PON 1 in the presence of clinical symptoms [ , ] and in patients with severe preeclampsia [ ].

    These results indicate consumption of antioxidants to combat heightened lipid peroxidation, which may injure vascular endothelium, and likely be involved in the pathogenesis of preeclampsia [ , ]. Affected women also have a decreased total antioxidant status TAS , placental GPx [ , , ], and low levels of vitamins C and E [ ]. Inadequate vitamin C intake seems to be associated with an increased risk of preeclampsia [ ] and some studies have shown that peri-conceptional supplementation with multivitamins may lower the risk of preeclampsia in normal or under-weight women [ , ].

    However, the majority of trials to date have found routine antioxidant supplementation during pregnancy to be ineffective in reducing the risk of preeclampsia [ , - ]. Intra uterine growth restriction is defined as infant birth weight below the 10 th percentile. Placental, maternal, and fetal factors are the most common causes of IUGR. Preeclampsia is an important cause of IUGR, as it develops from uteroplacental insufficiency and ischemic mechanisms in the placenta [ ]. Imbalanced injury and repair as well as abnormal development of the villous tree are characteristic of IUGR placentas, predisposing them to depletion of the syncytiotrophoblast with consequently limited regulation of transport and secretory function.

    Women with IUGR have been reported to have increased free radical activity and markers of lipid peroxidation [ ]. Furthermore, Biri et al reported that higher levels of MDA and xanthine oxidase and lower levels of antioxidant concentrations in the plasma, placenta, and umbilical cords in patients with IUGR compared to controls [ ]. Urinary 8-oxo-7,8- dihydrodeoxyguanosine 8-OxOdG , a marker of DNA oxidation, was also observed to be elevated at 12 and 28 weeks in pregnancies complicated with growth-restricted fetuses compared with a control group [ ].

    The regulatory apoptotic activity of p53 [ ] is significantly increased in response to hypoxic conditions within villous trophoblasts [ - ] and signifies a greater degree of apoptosis secondary to hypoxia-reoxygenation [ ] than from hypoxia alone [ ]. Furthermore, disordered protein translation and signaling in the placenta can also cause ER stress in the syncytiotrophoblast, and has been demonstrated in placentas of IUGR patients [ ].

    ER stress inhibits placental protein synthesis, eventually triggering apoptosis [ ]. Moreover, induction of p38 and NF-kappa B pathways can occur through ER stress, exacerbating inflammatory responses [ ]. The chronicity these events may explain the placental growth restriction seen in these pregnancies [ ]. In addition, serum prolidase activity in patients with IUGR was significantly elevated and negatively correlated with TAC, suggesting increased and dysregulated collagen turnover [ ]. The sequence of uterine contraction, cervical dilatation, and decidual activation make up the uterine component of this pathway [ ].

    However, it has been proposed that activation of this common pathway through physiological signals results in term labor, while preterm labor might occur from spontaneous activation of isolated aspects of the common pathway by the presence of pathological conditions that may be induced by multiple causes [ ] or risk factors.

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    Preterm labor in general is divided in two distinctive types: indicated , usually due to maternal or fetal reasons, or spontaneous. The majority of spontaneous preterm deliveries occur from any of the four primary pathogenic pathways. These include uterine overdistension, ischemia, infection, cervical disease, endocrine disorders [ ], decidual hemorrhage, and maternal-fetal activation of the hypothalamic-pituitary axis, amongst others [ ].

    Of these etiologies, intrauterine infection and inflammation is considered a main contributor to preterm birth [ ]. These pathogenic mechanisms converge on a common pathway involving increased protease expression and uterotonin. More than one process may take place in a given woman. The combination of genetics and inflammatory responses is an active area of research that could explain preterm labor in some women with common risk factors [ , ].

    Labor induces changes in chorioamniotic membranes that are consistent with localized acute inflammatory responses, despite the absence of histological evidence of inflammation [ ]. Reactive oxygen species activates NF-kappa B, which stimulates COX-2 expression and promotes inflammation with subsequent parturition. A study by Khan et al reported markedly decreased GPx protein expression in both women with preterm labor and those with term labor, compared with the respective non-labor groups [ ].

    Taken together, these data suggest that the state of labor, whether preterm or term, necessitates the actions of GPx to limit lipid oxidation, and is associated with an ROS-induced reduction of antioxidant defenses. Mustafa et al detected markedly higher levels of MDA and 8-OHdG and significantly lower GSH levels in the maternal blood of women with preterm labor than in women with term deliveries [ ]. This finding suggested that women in preterm labor have diminished antioxidant abilities to defend against OS-induced damage. The results further support that a maternal environment of increased OS and decreased antioxidants renders both the mother and fetus more susceptible to ROS-induced damage.

    Inflammation induces the up-regulation of ROS and can cause overt OS, resulting in tissue injury and subsequent preterm labor [ ]. Accordingly, higher mRNA expression of Mn-SOD was observed in the fetal membranes of women in preterm labor than in women in spontaneous labor at term, which may suggest a greater extent of OS and inflammatory processes in the former [ ]. Preterm labor has been associated with chorioamnionitis and histological infection was found to relate to elevated fetal membrane expression of Mn-SOD mRNA of women in preterm labor [ ]. Specifically, significantly higher amounts of the pro-inflammatory cytokines IL-1 beta, IL-6, and IL-8, have been observed in the amnion and choriodecidua of patients in preterm labor than in women in spontaneous term labor.

    These findings support activation of the membrane inflammatory response of women in preterm labor [ ]. Women with preterm labor have lower levels of TAS than women with uncomplicated pregnancies at a similar gestational age, which might indicate the presence of increased OS during preterm labor [ ]. Women with preterm births have also been found to have significantly decreased PON 1 activity in comparison to controls [ ]. This finding suggests that enhanced lipid peroxidation and diminished antioxidant activity of PON 1, may together create a pro-oxidant setting and increase the risk for preterm birth.

    Additionally, patients in preterm labor had markedly decreased levels of GSH [ ]. Low maternal serum selenium levels in early gestation have been associated with preterm birth [ ]. Polymorphism to GST was found to be significantly higher in patients in preterm labor, indicating that these patients are more vulnerable to oxidative damage [ ]. The inflammatory setting of maternal infection associated with preterm birth produces a state of OS and the consequent decrease in antioxidant defenses are likely to increase the risk for preterm birth. The presented evidence implicates inflammation and suppressed antioxidant defenses in the pathogenesis of preterm labor.

    Thus, it seems plausible that antioxidant supplementation may assist in preventing preterm labor and birth associated with inflammation. A study by Temma-Asano et al demonstrated that NAC was effective in reducing chorioamnionitis-induced OS, and thus, may protect against preterm labor [ ]. However, maternal supplementation with vitamins C and E in low-risk nulliparous patients during early gestation did not reduce preterm births [ , ].

    Due to the conflicting results of studies, it is unclear whether maternal antioxidant supplementation plays a role in preventing the onset of preterm labor. Pregnancy is a state of increased metabolic demands required to support both maternal hormonal physiology and normal fetal development. However, inadequate or excessive pregnancy weight gain can complicate both maternal and fetal health [ ]. The adverse effects of maternal obesity and underweight on fertility from disordered hormones and menses have been well-documented [ ].

    Ideally, women with a normal pre-pregnancy BMI Overweight women BMI Close to two-thirds of the United States population of reproductive-aged women are considered overweight or obese [ ]. Obese women generally take longer to conceive and have a higher risk of miscarriage than their leaner counterparts [ ]. Maternal obesity has also long been associated with several reproductive pathologies including gestational diabetes mellitus, preeclampsia, and PCOS.

    It has also been shown to negatively affect fertility and pregnancy. Healthy pregnancies are associated with the mobilization of lipids, increased lipid peroxides, insulin resistance, and enhanced endothelial function. Normally, increases in total body fat peak during the 2 nd trimester. Obese women, however, experience inappropriately increased lipid peroxide levels and limited progression of endothelial function during their pregnancies, along with an additive innate tendency for central fat storage.

    Visceral fat is associated with disordered metabolism and adipokine status, along with insulin resistance. Centrally-stored fat deposits are prone to fatty acid overflow, thereby exerting lipotoxic effects on female reproductive ability [ ].

    What is Oxidative Stress, Free Radicals & Antioxidants - Katie Rose

    Oxidative stress from excessive ROS generation has been implicated in pathogenesis of obesity [ ]. Intracellular fat accumulation can disrupt mitochondrial function, causing buildup and subsequent leak of electrons from the ETC. The combined effect of high lipid levels and OS stimulates production of oxidized lipids; of particular importance are lipid peroxides, oxidized lipoproteins, and oxysterols.

    A tutorial on oxidative stress and redox signaling with application to exercise and sedentariness

    As major energy producers for cells, the mitochondria synthesize ATP via oxidative phosphorylation. Adverse effects of maternal BMI on mitochondria in the oocyte could negatively influence embryonic metabolism. Increased plasma non-esterified fatty acid levels can prompt the formation of the nitroxide radical. As a known inflammatory mediator, oxLDL can indirectly measure lipid-induced OS, hence elucidating its role in the inflammatory state of obesity [ ].

    Oxysterol production within a lipotoxic environment can potentially disrupt the placental development and function of obese pregnancies [ ]. Consumption of a high fat meal has been shown to increase levels of both circulating endotoxins and markers of endothelial dysfunction [ - ]. Extensive evidence has linked endothelial dysfunction, increased vascular endothelial cell expression of NADPH oxidase, and endothelial OS to obesity. Overactive mitochondria and harmful ROS levels in oocytes and zygotes were influenced by peri-conceptional maternal obesity.

    Igosheva et al reported a decline in fertility and obscured progression of the developing embryo [ ]. The correlation between placental nitrative stress from altered vascular endothelial NO release and high maternal BMI [ ] may stem from imbalances of oxidative and nitrative stress, which may weaken protection to the placenta [ ]. Results from Ruder et al supported the association of increased maternal body weight and increased nitrative stress, but did not demonstrate a relation to placental OS [ 4 ]. Overabundant nutrition may produce an unfavorably rich reproductive environment, leading to modified oocyte metabolism and hindered embryo development.

    A negative association was also made between maternal diet-induced obesity and blastocyst development [ ]. Increased postprandial levels of OS biomarkers have been described after ingestion of high fat meals. A study by Bloomer et al found a greater increase in postprandial MDA in obese females versus normal weight controls [ ]. Hallmark events of obese states include decreased fatty acid uptake, enhanced lipolysis, infiltration of inflammatory cells, and secretion of adipokines [ , ]. Suboptimal oocyte quality has also been noted in obese females. The resultant disturbance of oocyte development may influence oocyte quality and perhaps general ovarian function.

    Maternal obesity has been linked to several increased risks to the mother, embryo, and fetus. Obesity is considered a modifiable risk factor; therefore, pre-conceptional counseling should stress the importance of a balanced diet and gestational weight gain within normal limits. Nutritional deficiencies in underdeveloped areas of the world continue to be a significant public health concern. Inadequate maternal nutrition during the embryonic period adversely affects fetal growth, placing a pregnant woman at risk for a low birth weight infant and potential endothelial dysfunction.

    Malnourished females and those with a low BMI may be at increased risk for impaired endothelium-dependent vasodilation secondary to OS [ ]. In-utero undernutrition reduces NO stores, triggering OS along with impairment of endothelium-dependent vasodilation.

    In rodents, gestational exposure to both caloric and protein restriction resulted in low birth weight offspring. The activity of SOD was found to be decreased with a consequent increase of the SO anion in the offspring of undernourished dams, which also indicates decreased formation of H 2 O 2. Elevated SO anion levels also stimulate NO scavenging and cell damage associated with endothelial dysfunction [ ]. Primordial, secondary, and antral follicle numbers markedly decrease in relation to time intervals of limited nutritional exposure.

    Insufficient maternal nutrition, especially during critical periods of embryonic and fetal development, manifests as an overall elevation of ovarian OS, which, along with impaired mitochondrial antioxidant defenses, may be responsible for these significantly decreased follicle numbers and resultant growth impediment of offspring [ ]. In general, adolescence is a period of increased physiological demands for growth and development. If a pregnancy occurs during this time, it creates an environment in which mother and fetus compete for nutrients, as both parties are undergoing major developmental changes throughout gestation.

    Inadequate nutrition during adolescence is especially problematic, as youths often lack one or more vital micronutrients. Given the varied requirements of different communities and populations for health maintenance, antioxidant or mineral supplementation should be population-specific. Physical exercise produces an oxidative state due to excessive ROS generation.

    Any type of extreme aerobic or anaerobic activity e. Optimal amounts of OS are necessary for physiologic functioning. Physical activity causes an increase in ROS, which in turn heightens antioxidant response, thus providing protection from future attacks [ ].

    An overproduction of OS after acute exercise in certain diseased individuals may serve as a trigger for improved antioxidant defense when compared with their healthy counterparts [ ]. Leelarungrayub et al established that aerobic exercise can increase TAC and decrease MDA levels, resulting in better physical fitness in previously sedentary women [ ]. Maternal BMI has great potential to affect pregnancy outcomes and would likely benefit from further research.

    The 21st century has been burdened with a sharp increase in the use of several substances of abuse. This problem significantly affects the younger generations, which encompass the female reproductive years. Cigarette smoking, alcohol use, and recreational drug use have been implicated in the pathogenesis of perturbed female reproductive mechanisms, leading to increased times to conception and infertility [ ]. The nicotine component of cigarette smoke is notoriously addictive and toxic to the human body.

    In the United States, approximately one-third of women in the reproductive age group smoke cigarettes. Maternal smoking is associated with infertility, pregnancy complications, and damage to the developing embryo. Higher rates of fetal loss, decreased fetal growth [ ], and preterm birth have also been associated with maternal smoking. The risk of spontaneous abortion has been found to be greatly increased in smokers versus non-smokers [ ].