Nevertheless, in the case of 3,5-DCA, there was no significant increase in the GSSG/GSH ratio, and the significant increase in protein carbonyl levels only occurred after there was an increase in cytotoxicity. a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, -tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be triggered via several renal enzyme systems to harmful metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro. and (Hong et al., 1997; Rankin et al., 1994, 2008a; Valentovic et al., 1997). Interestingly, addition of a chloro group to the 4-position of 3,5-DCA to form 3,4,5-trichloroaniline generates a 3,5-DCA derivative without the ability to form significant amounts of 4-amino-2,6-dichlorophenol. However, 3,5-DCA and 3,4,5-trichloroaniline have equivalent nephrotoxic potential at 90 min, and 3,4,5-trichloroaniline is definitely more potent like a nephrotoxicant than 3,5-DCA at 120min in IRCC (Racine et al., 2014). Therefore, although 4-amino-2,6-dichlorophenol is definitely a nephrotoxicant, it does not look like the ultimate nephrotoxic metabolite arising from 3,5-DCA in vitro. Studies with 2-amino-4,6-dichlorophenol are ongoing to determine its nephrotoxic potential. Therefore, the part of aminophenol metabolites in 3,5-DCA cytotoxicity remains to be fully identified, but oxidation in the 4-position of 3,5-DCA does not look like a critical bioactivation pathway. Since the general CYP inhibitors (piperonyl butoxide and metyrapone) were able to significantly attenuate cytotoxicity, further studies were conducted looking at the part of selective CYP isozymes which are found in the kidney. Cummings et al. (1999) found out CYP2E1, CYP2C11, CYP2B1/2, and CYP4A2/3 in freshly isolated rat proximal and distal tubular cells. CYP2E1 manifestation was higher in distal tubular cells than proximal tubular cells, while CYP2C11 was higher in proximal tubular cells than distal tubular cells. CYP3A1/2 was not recognized in the proximal tubular cells but was found in total kidney homogenate, which may indicate why oleandomycin, a CYP3A inhibitor, was not effective in attenuating 3,5-DCA cytotoxicity. The inability of thio-tepa (CYP2B inhibitor) and isoniazid (CYP2E inhibitor) to attenuate 3,5-DCA cytotoxicity, suggests that these CYPs are not critical for 3,5-DCA bioactivation. Of the selective CYP inhibitors we used, only sulfaphenazole, omeprazole, and diethyldithiocarbamate (DEDTCA) were able to attenuate 3,5-DCA cytotoxicity. These three inhibitors all display a preference to inhibit the 2C family of rat isozymes (Eagling et al., 1998; Kobayashi et al., 2003), suggesting the 2C family may play a role in the bioactivation of 3,5-DCA. The CYP2C family in rats facilitates N-hydroxylation, as well as aromatic ring oxidation (Cribb et al., 1995), YO-01027 which helps one or both of these pathways as contributing to 3,5-DCA bioactivation. Both N-hydroxylation and aromatic ring oxidation can lead to an increase in free radicals: either as metabolites undergoing redox cycling or directly from oxidation during rate of metabolism (Harmon et al., 2006; Michail et al., 2013), and N-hydroxyl, N-nitroso and aminophenol metabolites can induce cell death via oxidative stress mechanisms (Harmon et al., 2005; Lock et al., 1993; Umbreit, 2007; Valentovic et al., 1997). Antioxidant pretreatment proved to be highly effective in attenuating 3,5-DCA cytotoxicity, with all antioxidants offering protection, suggesting that free radicals may play a role in cytotoxicity. Oxidative stress was measured by looking in the percentage of GSSG/GSH and raises in protein carbonyl levels. If oxidative stress played a significant part in the mechanism of cellular death, an increase in the GSSG/GSH percentage should happen prior to cytotoxicity, as seen with compounds such as em virtude de-aminophenol (Harmon et al., 2005). However, in the case of 3,5-DCA, there was no significant increase in the GSSG/GSH percentage, and the significant increase in protein carbonyl levels only occurred after there was an increase in cytotoxicity. These data suggest that oxidative stress is not responsible for cell death in 3,5-DCA induced nephrotoxicity in vitro, and that the antioxidants may be providing security, at least partly, by scavenging a number of radical metabolites created during the fat burning capacity from the amino group or an aminophenol metabolite (e.g. 2-amino-4,6-dichlorophenol) (Fowler et al.,1991, 1993; Work et al., 1978). Total glutathione amounts decreased pursuing 3,5-DCA publicity, despite the fact that the GSSG/GSH proportion didnt significantly modification (Fig. 7). In the lack of oxidative tension, chances are that the decrease in total glutathione amounts was because of the formation of the reactive 3,5-DCA metabolite(s) that was(had been) detoxified by response with minimal glutathione. Both addition of N-acetyl-L-cysteine and GSH, which secure cells when you are changed into GSH (Lauterburg et al. 1983), to IRCC attenuated 3,5-DCA cytotoxicity. Hence, at least component.From the selective CYP inhibitors we used, only sulfaphenazole, omeprazole, and diethyldithiocarbamate (DEDTCA) could actually attenuate 3,5-DCA cytotoxicity. but 120 min was necessary for 3,5-DCA 0.5 mM to improve LDH discharge. In subsequent research, IRCC were subjected to a pretreatment (antioxidant or enzyme inhibitor) ahead of contact with 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Usage of even more selective CYP inhibitors recommended the fact that CYP 2C family members added to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, -tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione amounts as well as the oxidized/decreased glutathione ratios weren’t increased. These outcomes indicate that 3,5-DCA could be turned on via many renal enzyme systems to poisonous metabolites, which free radicals, however, not oxidative tension, donate to 3,5-DCA induced nephrotoxicity in vitro. and (Hong et al., 1997; Rankin et al., 1994, 2008a; Valentovic et al., 1997). Oddly enough, addition of the chloro group towards the 4-placement of 3,5-DCA to create 3,4,5-trichloroaniline creates a 3,5-DCA derivative without the capability to form quite a lot of 4-amino-2,6-dichlorophenol. Nevertheless, 3,5-DCA and 3,4,5-trichloroaniline possess similar nephrotoxic potential at 90 min, and 3,4,5-trichloroaniline is certainly more potent being a nephrotoxicant than 3,5-DCA at 120min in IRCC (Racine et al., 2014). Hence, although 4-amino-2,6-dichlorophenol is certainly a nephrotoxicant, it generally does not seem to be the best nephrotoxic metabolite due to 3,5-DCA in vitro. Research with 2-amino-4,6-dichlorophenol are ongoing to determine its nephrotoxic potential. Hence, the function of aminophenol metabolites in 3,5-DCA cytotoxicity continues to be to be completely motivated, but oxidation on the 4-placement of 3,5-DCA will not seem to be a crucial bioactivation pathway. Because the general CYP inhibitors (piperonyl butoxide and metyrapone) could actually considerably attenuate cytotoxicity, further research were conducted taking a look at the function of selective CYP isozymes which are located in the kidney. Cummings et al. (1999) present CYP2E1, CYP2C11, CYP2B1/2, and CYP4A2/3 in newly isolated rat proximal and distal tubular cells. CYP2E1 appearance was higher in distal tubular cells than proximal tubular cells, while CYP2C11 was higher in proximal tubular cells than distal tubular cells. CYP3A1/2 had not been discovered in the proximal tubular cells but was within total kidney homogenate, which might indicate why oleandomycin, a CYP3A inhibitor, had not been effective in attenuating 3,5-DCA cytotoxicity. The shortcoming of thio-tepa (CYP2B inhibitor) and isoniazid (CYP2E inhibitor) to attenuate 3,5-DCA cytotoxicity, shows that these CYPs aren’t crucial for 3,5-DCA bioactivation. From the selective CYP inhibitors we utilized, just sulfaphenazole, omeprazole, and diethyldithiocarbamate (DEDTCA) could actually attenuate 3,5-DCA cytotoxicity. These three inhibitors all present a choice to inhibit the 2C category of rat isozymes (Eagling et al., 1998; Kobayashi et al., 2003), recommending the fact that 2C family members may are likely involved in the bioactivation of 3,5-DCA. The CYP2C family members in rats facilitates N-hydroxylation, aswell as aromatic band oxidation (Cribb et al., 1995), which works with one or both these pathways as adding to 3,5-DCA bioactivation. Both N-hydroxylation and aromatic band oxidation can result in a rise in free of charge radicals: either as metabolites going through redox bicycling or straight from oxidation during fat burning capacity (Harmon et al., 2006; Michail et al., 2013), and N-hydroxyl, N-nitroso and aminophenol metabolites can induce cell loss of life via oxidative tension systems (Harmon et al., 2005; Lock et al., 1993; Umbreit, 2007; Valentovic et al., 1997). Antioxidant pretreatment became impressive in attenuating 3,5-DCA cytotoxicity, with all antioxidants providing protection, recommending that free of charge radicals may are likely involved in cytotoxicity. Oxidative tension was assessed by looking in the percentage of GSSG/GSH and raises in proteins carbonyl amounts. If oxidative tension played a substantial part in the system of cellular loss of life, a rise in the GSSG/GSH percentage should occur ahead of cytotoxicity, as noticed with compounds such as for example em virtude de-aminophenol (Harmon et al., 2005). Nevertheless, regarding 3,5-DCA, there is no significant upsurge in the GSSG/GSH percentage, as well as the significant upsurge in proteins carbonyl amounts only happened after there is a rise in cytotoxicity. These data claim that oxidative tension is not in charge of cell loss of life in 3,5-DCA induced nephrotoxicity in vitro, which the antioxidants could be providing safety, at least partly, by scavenging a number of radical metabolites created during the rate of metabolism from the amino group or an aminophenol metabolite (e.g. 2-amino-4,6-dichlorophenol) (Fowler et al.,1991, 1993; Work et al., 1978). Total glutathione amounts decreased pursuing 3,5-DCA publicity, despite the fact that the GSSG/GSH percentage didnt significantly modification (Fig. 7). In the lack of oxidative tension, chances are that the decrease in total glutathione amounts was because of the formation of the reactive 3,5-DCA metabolite(s) that was(had been) detoxified by response with minimal glutathione. Both addition of GSH and N-acetyl-L-cysteine, which shield cells when you are changed into GSH (Lauterburg et.2-amino-4,6-dichlorophenol) (Fowler et al.,1991, 1993; Work et al., 1978). Total glutathione levels reduced subsequent 3,5-DCA exposure, despite the fact that the GSSG/GSH percentage didnt significantly modification (Fig. subjected to a pretreatment (antioxidant or enzyme inhibitor) ahead of contact with 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Usage of even more selective CYP inhibitors recommended how the CYP 2C family members added to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, -tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione amounts as well as the oxidized/decreased glutathione ratios weren’t increased. These outcomes indicate that 3,5-DCA could be triggered via many renal enzyme systems to poisonous metabolites, which free radicals, however, not oxidative tension, donate to 3,5-DCA induced nephrotoxicity in vitro. and (Hong et al., 1997; Rankin et al., 1994, 2008a; Valentovic et al., 1997). Oddly enough, addition of the chloro group towards the 4-placement of 3,5-DCA to create 3,4,5-trichloroaniline generates a 3,5-DCA derivative without the capability to form quite a lot of 4-amino-2,6-dichlorophenol. Nevertheless, 3,5-DCA and 3,4,5-trichloroaniline possess similar nephrotoxic potential at 90 min, and 3,4,5-trichloroaniline can be more potent like a nephrotoxicant than 3,5-DCA at 120min in IRCC (Racine et al., 2014). Therefore, although 4-amino-2,6-dichlorophenol can be a nephrotoxicant, it generally does not look like the best nephrotoxic metabolite due to 3,5-DCA in vitro. Research with 2-amino-4,6-dichlorophenol are ongoing to determine its nephrotoxic potential. Therefore, the part of aminophenol metabolites in 3,5-DCA cytotoxicity continues to be to be completely established, but oxidation in the 4-placement of 3,5-DCA will not look like a crucial bioactivation pathway. Because the general CYP inhibitors (piperonyl butoxide and metyrapone) could actually considerably attenuate cytotoxicity, further research were conducted taking a look at the part of selective CYP isozymes which are located in the kidney. Cummings et al. (1999) found out CYP2E1, CYP2C11, CYP2B1/2, and CYP4A2/3 in newly isolated rat proximal and distal tubular cells. CYP2E1 manifestation was higher in distal tubular cells than proximal tubular cells, while CYP2C11 was higher in proximal tubular cells than distal tubular cells. CYP3A1/2 had not been recognized in the proximal tubular cells but was within total kidney homogenate, which might indicate why oleandomycin, a CYP3A inhibitor, had not been effective in attenuating 3,5-DCA cytotoxicity. The shortcoming of thio-tepa (CYP2B inhibitor) and isoniazid (CYP2E inhibitor) to attenuate 3,5-DCA cytotoxicity, shows that these CYPs aren’t crucial for 3,5-DCA bioactivation. From the selective CYP inhibitors we utilized, just sulfaphenazole, omeprazole, and diethyldithiocarbamate (DEDTCA) could actually attenuate 3,5-DCA cytotoxicity. These three inhibitors all display a choice to inhibit the 2C category of rat isozymes (Eagling et al., 1998; Kobayashi et al., 2003), recommending how the 2C family members may are likely involved in the bioactivation of 3,5-DCA. The CYP2C family members in rats facilitates N-hydroxylation, aswell as aromatic band oxidation (Cribb et al., 1995), which helps one or both these pathways as adding to 3,5-DCA bioactivation. Both N-hydroxylation and aromatic band oxidation can result in a rise in free of charge radicals: either as metabolites going through redox bicycling or straight from oxidation during fat burning capacity (Harmon et al., 2006; Michail et al., 2013), and N-hydroxyl, N-nitroso and aminophenol metabolites can induce cell loss of life via oxidative tension systems (Harmon et al., 2005; Lock et al., 1993; Umbreit, 2007; Valentovic et al., 1997). Antioxidant pretreatment became impressive in attenuating 3,5-DCA cytotoxicity, with all antioxidants providing protection, recommending that free of charge radicals may are likely involved in cytotoxicity. Oxidative tension was assessed by looking on the proportion of GSSG/GSH and boosts in proteins carbonyl amounts. If oxidative tension played a substantial function in the system of cellular loss of life, a rise in the GSSG/GSH proportion should occur ahead of cytotoxicity, as noticed with compounds such as for example em fun??o de-aminophenol (Harmon et al., 2005). Nevertheless, regarding 3,5-DCA, there is no significant upsurge in the GSSG/GSH proportion, as well as the significant upsurge in proteins carbonyl amounts only happened after there is a rise in cytotoxicity. These data claim that oxidative tension is not in charge of cell loss of life in 3,5-DCA induced nephrotoxicity in vitro, which the antioxidants could be providing security, at least partly, by scavenging a number of radical metabolites created during the fat burning capacity from the amino group or an aminophenol metabolite (e.g. 2-amino-4,6-dichlorophenol) (Fowler et al.,1991, 1993; Work et al., 1978). Total glutathione amounts decreased pursuing 3,5-DCA publicity, despite the fact that the GSSG/GSH proportion didnt significantly transformation (Fig. 7). In the lack of oxidative tension, chances are that the decrease in total glutathione amounts was because of the development of.If oxidative tension played a substantial function in the system of cellular loss of life, a rise in the GSSG/GSH proportion should occur ahead of cytotoxicity, as noticed with compounds such as for example em fun??o de-aminophenol (Harmon et al., 2005). P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Usage of even more selective CYP inhibitors recommended which the CYP 2C family members added to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, -tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione amounts as well as the oxidized/decreased glutathione ratios weren’t increased. These outcomes indicate that 3,5-DCA could be turned on via many renal enzyme systems to dangerous metabolites, which free radicals, however, not oxidative tension, donate to 3,5-DCA induced nephrotoxicity in vitro. and (Hong et al., 1997; Rankin et al., 1994, 2008a; Valentovic et al., 1997). Oddly enough, addition of the chloro group towards the 4-placement of 3,5-DCA to create 3,4,5-trichloroaniline creates a 3,5-DCA derivative without the capability to form quite a lot of 4-amino-2,6-dichlorophenol. Nevertheless, 3,5-DCA and 3,4,5-trichloroaniline possess equivalent nephrotoxic potential at 90 min, and 3,4,5-trichloroaniline is usually more potent as a nephrotoxicant than 3,5-DCA at 120min in IRCC (Racine et al., 2014). Thus, although 4-amino-2,6-dichlorophenol is YO-01027 usually a nephrotoxicant, it does not appear to be the ultimate nephrotoxic metabolite arising from 3,5-DCA in vitro. Studies with 2-amino-4,6-dichlorophenol are ongoing to determine its nephrotoxic potential. Thus, the role of aminophenol metabolites in 3,5-DCA cytotoxicity remains to be fully decided, but oxidation at the 4-position of 3,5-DCA does not appear to be a critical bioactivation pathway. Since the general CYP inhibitors (piperonyl butoxide and metyrapone) were able to significantly attenuate cytotoxicity, further studies were conducted looking at the role of selective CYP isozymes which are found in the kidney. Cummings et al. (1999) found CYP2E1, CYP2C11, CYP2B1/2, and CYP4A2/3 in freshly isolated rat proximal and distal tubular cells. CYP2E1 expression was higher in distal tubular cells than proximal tubular cells, while CYP2C11 was higher in proximal tubular cells than distal tubular cells. CYP3A1/2 was not detected in the proximal tubular cells but was found in total kidney homogenate, which may indicate why oleandomycin, a CYP3A inhibitor, was not effective in attenuating 3,5-DCA cytotoxicity. The inability of thio-tepa (CYP2B inhibitor) and isoniazid (CYP2E inhibitor) to attenuate 3,5-DCA cytotoxicity, suggests that these CYPs are not critical for 3,5-DCA bioactivation. Of the selective CYP inhibitors we used, only sulfaphenazole, omeprazole, and diethyldithiocarbamate (DEDTCA) were able to attenuate 3,5-DCA cytotoxicity. These three inhibitors all show a preference to inhibit the 2C family of rat isozymes (Eagling et al., 1998; Kobayashi et al., 2003), suggesting that this 2C family may play a role in the bioactivation of 3,5-DCA. The CYP2C family in rats facilitates N-hydroxylation, as well as aromatic ring oxidation (Cribb et al., 1995), which supports one or both of these pathways as contributing to 3,5-DCA bioactivation. Both N-hydroxylation and aromatic ring oxidation can lead to an increase in free radicals: either as metabolites undergoing redox cycling or directly from oxidation during metabolism (Harmon et al., 2006; Michail et al., 2013), and N-hydroxyl, N-nitroso and aminophenol metabolites can induce cell death via oxidative stress mechanisms (Harmon et al., 2005; Lock et al., 1993; Umbreit, 2007; Valentovic et al., 1997). Antioxidant pretreatment proved to be highly effective in attenuating 3,5-DCA cytotoxicity, with all antioxidants offering protection, suggesting that free radicals may play a role in cytotoxicity. Oxidative stress was measured by looking at the ratio of GSSG/GSH and increases in protein carbonyl levels. If oxidative stress played a significant role in the mechanism of cellular death, an increase in the GSSG/GSH ratio should occur prior to cytotoxicity, as seen with compounds such as para-aminophenol.The inability TTK of thio-tepa (CYP2B inhibitor) and isoniazid (CYP2E inhibitor) to attenuate 3,5-DCA cytotoxicity, suggests that these CYPs are not critical for 3,5-DCA bioactivation. but 120 min was required for 3,5-DCA 0.5 mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that this CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, -tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to harmful metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro. and (Hong et al., 1997; Rankin et al., 1994, 2008a; Valentovic et al., 1997). Interestingly, addition of a chloro group to the 4-position of 3,5-DCA to form 3,4,5-trichloroaniline produces a 3,5-DCA derivative without the ability to form significant amounts of 4-amino-2,6-dichlorophenol. However, 3,5-DCA and 3,4,5-trichloroaniline have equivalent nephrotoxic potential at 90 min, and 3,4,5-trichloroaniline is usually more potent as a nephrotoxicant than 3,5-DCA at 120min in IRCC (Racine et al., 2014). Thus, although 4-amino-2,6-dichlorophenol is usually a nephrotoxicant, it does not appear to be the ultimate nephrotoxic metabolite arising from 3,5-DCA in vitro. Studies with 2-amino-4,6-dichlorophenol are ongoing to determine its nephrotoxic potential. Thus, the role of aminophenol metabolites in 3,5-DCA cytotoxicity remains to be fully decided, but oxidation at the 4-position of 3,5-DCA does not appear to be a critical bioactivation pathway. Since the general CYP inhibitors (piperonyl butoxide and metyrapone) were able to significantly attenuate cytotoxicity, further studies were conducted looking at the role of selective CYP isozymes which are found in the kidney. Cummings et al. (1999) found CYP2E1, CYP2C11, CYP2B1/2, and CYP4A2/3 in freshly isolated rat proximal and distal tubular cells. CYP2E1 expression was higher in distal tubular cells than proximal tubular cells, while CYP2C11 was higher in proximal tubular cells than distal tubular cells. CYP3A1/2 was not detected in the proximal tubular cells but was found in total kidney homogenate, which may indicate why oleandomycin, a CYP3A inhibitor, was not effective in attenuating 3,5-DCA cytotoxicity. The YO-01027 inability of thio-tepa (CYP2B inhibitor) and isoniazid (CYP2E inhibitor) to attenuate 3,5-DCA cytotoxicity, suggests that these CYPs are not critical for 3,5-DCA bioactivation. Of the selective CYP inhibitors we used, only sulfaphenazole, omeprazole, and diethyldithiocarbamate (DEDTCA) were able to attenuate 3,5-DCA cytotoxicity. These three inhibitors all show a preference to inhibit the 2C family of rat isozymes (Eagling et al., 1998; Kobayashi et al., 2003), suggesting that the 2C family may play a role in the bioactivation of 3,5-DCA. The CYP2C family in rats facilitates N-hydroxylation, as well as aromatic ring oxidation (Cribb et al., 1995), which supports one or both of these pathways as contributing to 3,5-DCA bioactivation. Both N-hydroxylation and aromatic ring oxidation can lead to an increase in free radicals: either as metabolites undergoing redox cycling or directly from oxidation during metabolism (Harmon et al., 2006; Michail et al., 2013), and N-hydroxyl, N-nitroso and aminophenol metabolites can induce cell death via oxidative stress mechanisms (Harmon et al., 2005; Lock et al., 1993; Umbreit, 2007; Valentovic et al., 1997). Antioxidant pretreatment proved to be highly effective in attenuating 3,5-DCA cytotoxicity, with all antioxidants offering protection, suggesting that free radicals may play a role in cytotoxicity. Oxidative stress was measured by looking at the ratio of GSSG/GSH and increases in protein carbonyl levels. If oxidative stress played a significant role in the mechanism of cellular death, an increase in the GSSG/GSH ratio should occur prior to cytotoxicity, as seen with compounds such as para-aminophenol (Harmon et al., 2005). However, in the case of 3,5-DCA, there was no significant increase in the GSSG/GSH ratio, and the significant increase in protein carbonyl levels only occurred after there was an increase in cytotoxicity. These data suggest that oxidative stress is not responsible for cell death in 3,5-DCA induced nephrotoxicity in vitro, and that the antioxidants may be offering protection, at least in part, by scavenging one or more radical metabolites produced during the metabolism of the amino group or an aminophenol metabolite (e.g. 2-amino-4,6-dichlorophenol) (Fowler et al.,1991, 1993; Job et al., 1978). Total glutathione levels decreased following 3,5-DCA exposure, even though the GSSG/GSH ratio didnt significantly change (Fig. 7). In the absence of oxidative stress, it is likely that the reduction in total glutathione levels was due to the formation of a reactive 3,5-DCA metabolite(s) that was(were) detoxified by reaction with reduced glutathione. Both addition of GSH and N-acetyl-L-cysteine, which protect.