CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes

Cytochrome P4502E1 (CYP2E1), glutathione-S-transferase A4-4 (GSTA4-4), and inducible nitric oxide synthase (iNOS) are implicated in maneb- and paraquat-induced toxicity leading to various pathological conditions. The study aimed to investigate the role of CYP2E1 in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) and its crosstalk with iNOS-mediated nitrosative stress and GSTA4-4-linked protective effect, if any and their consequent links with the nuclear factor erythoid 2-related factor 2 (Nrf2) activation and heme oxygenase-1 (HO-1) expression. Rats were treated with/without maneb and/or paraquat for 1, 2, and 3 weeks along with vehicle controls. Subsets of rats were also treated with diallyl sulfide (DAS) or aminoguanidine (AG) along with the respective controls. Maneb and paraquat augmented the reactive oxygen species (ROS), lipid peroxidation (LPO) and 4-hydroxy nonenal (4-HNE) contents, and superoxide dismutase (SOD) activity in the PMNs. However, maneb and paraquat attenuated the reduced glutathione (GSH) level and the expression/activity of total GST and GST-pi. Maneb and paraquat increased the expression/activity of CYP2E1, GSTA4-4, iNOS, Nrf2 and HO-1, and nitrite content. CYP2E1 inhibitor, DAS noticeably alleviated maneb- and paraquat-induced ROS, LPO, 4-HNE, SOD, Nrf2 and HO-1, GST, GSH, and GST-pi while iNOS, nitrite content and GSTA4-4 levels were unchanged. Conversely, AG, an iNOS inhibitor, attenuated maneb- and paraquat-directed changes in nitrite, LPO, iNOS but it did not alter ROS, GSH, SOD, GST, GST-pi, Nrf2, HO-1, CYP2E1, and GSTA4-4. The results demonstrate that CYP2E1 induces iNOS-independent free radical generation and subsequently modulates the Nrf2-dependent HO-1 and 4-HNE-mediated GST expression in maneb- and paraquat-treated PMNs.     

N-acetylcysteine pretreatment attenuates paraquat-induced lung leak in rats

We investigated the effects of N-acetylcysteine (NAC) pretreatment on paraquat-induced lung inflammation and leak. We found that administering a single intravenous dose (60 mg/kg) of paraquat rapidly (2 h) increased lung leak, lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, and lung myeloperoxidase (MPO) activity in rats. Rats pretreated with NAC (150 mg/kg, intraperitoneally) had increased lung tissue glutathione (GSH + GSSG) levels compared to saline-pretreated rats. In addition, rats pretreated with NAC and then given paraquat 2.5 h later had decreased lung leak compared to saline-pretreated rats given paraquat. In contrast, NAC pretreated rats given paraquat had the same lung lavage CINC levels and lung tissue MPO activity as saline-pretreated rats given paraquat. Our results indicate that paraquat causes an oxidative injury which may be decreased by the GSH-increasing or other properties of NAC.     

Co-Administration of Resveratrol and Lipoic Acid,or Their Synthetic Combination,Enhances Neuroprotection in a Rat Model of Ischemia/Reperfusion

The present study demonstrates the benefits of combinatorial antioxidant therapy in the treatment of ischemic stroke. Male Sprague-Dawley rats were anaesthetised and the middle cerebral artery (MCA) was occluded for 30 minutes followed by 5.5 hours of reperfusion. Pretreatment with resveratrol 30 minutes prior to MCA occlusion resulted in a significant, dose-dependent decrease in infarct volume (p<0.05) compared to vehicle-treated animals. Neuroprotection was also observed when resveratrol (2×10⁻³ mg/kg; iv) was administered within 60 minutes following the return of blood flow (reperfusion). Pretreatment with non-neuroprotective doses of resveratrol (2×10⁻⁶ mg/kg) and lipoic acid (LA; 0.005 mg/kg) in combination produced significant neuroprotection as well. This neuroprotection was also observed when resveratrol and LA were administered 15 minutes following the onset of MCA occlusion. Subsequently, we synthetically combined resveratrol and LA in both a 1∶3 (UPEI-200) and 1∶1 (UPEI-201) ratio, and screened these new chemical entities in both permanent and transient ischemia models. UPEI-200 was ineffective, while UPEI-201 demonstrated significant, dose-dependent neuroprotection. These results demonstrate that combining subthreshold doses of resveratrol and LA prior to ischemia-reperfusion can provide significant neuroprotection likely resulting from concurrent effects on multiple pathways. The additional protection observed in the novel compound UPEI 201 may present opportunities for addressing ischemia-induced damage in patients presenting with transient ischemic episodes.     

Paraquat detoxicative system in the mouse liver postmitochondrial fraction

We examined the paraquat detoxicative system in mouse livers. The survival rate of mice receiving 50 mg/kg paraquat was 41% at 7 days and significantly rose to 88, 64, 69% with pretreatment with phenytoin, phenobarbital, and rifampicin, respectively. Phenytoin induced activity in NADPH-cytochrome P450 reductase, CYP3A, CYP2B, and CYP2C that was 3 to 4 times higher than that of the controls. Phenobarbital induced CYP2B and rifampicin induced CYP3A, respectively, in addition to NADPH-cytochrome P450 reductase. 3-Methylcholanthrene did not induce these enzymes and did not alter the survival rate. All the mice pretreated with CoCl(2) (a CYP synthesis inhibitor) or SKF 525-A (a CYP inhibitor) were dead after 5 days, and troleandomycin (a CYP3A-specific inhibitor) also reduced the survival rate. When cell homogenates were incubated with paraquat and NADPH, paraquat decreased and its metabolic intermediate paraquat-monopyridone was formed. Troleandomycin inhibited the decrease in paraquat and increased the monopyridone. After making a subfraction of the homogenate, monopyridone was produced in the postmicrosomal 105,000g supernatant, but not in the microsomes. The pretreatment of mice with phenytoin decreased the monopyridone in the postmitochondrial fraction, but did not affect the supernatant. These results indicated that paraquat was first metabolized in the postmicrosomal supernatant into monopyridone, and that may have been subsequently hydroxylated by the microsomes. Repeated intravenous injections of alpha-tocopherol to paraquat-loaded mice significantly reduced the paraquat mortality and when these mice were pretreated with rifampicin, 100% of them survived. These studies demonstrate that postmitochondrial fractions play an important role in paraquat detoxication metabolism, and that the combination of CYP induction and alpha-tocopherol administration is highly useful for the survival of paraquat-exposed mice.     

Genotoxicity of paraquat: micronuclei induced in bone marrow and peripheral blood are inhibited by melatonin

The ability of melatonin to influence paraquat-induced genotoxicity was tested using micronucleated polychromatic erythrocytes as an index of damage in both bone marrow and peripheral blood cells of mice. Melatonin (10 mg/kg) or an equal volume of saline were administered intraperitoneally (ip) to mice 30 min prior to an ip injection of paraquat (20 mg/kgx2), and thereafter at 6-h intervals until the conclusion of the study (72 h). The number of the micronucleated polychromatic erythrocytes increased after paraquat administration both in peripheral blood and bone marrow cells. Melatonin administration to paraquat-treated mice significantly reduced micronuclei formation in both peripheral blood and bone marrow cells; these differences were apparent at 24, 48 and 72 h after paraquat administration. The induction of micronuclei was time-dependent with peak values occurring at 24 and 48 h. The reduction in paraquat-related genotoxicity by melatonin is likely due in part to the antioxidant activity of the indole. We did not observe effects of melatonin over paraquat in paraquat+melatonin groups incubated at 0, 60 and 120 min. Mitomycin C, which was used as a positive control, also caused the expected large rises in micronuclei in both bone marrow and peripheral blood cells at 24, 48 and 72 h after its administration.     

Cyp1a2 protects against reactive oxygen production in mouse liver microsomes

H(2)O(2) production was evaluated in liver microsomes prepared from Cyp1a1/1a2(+/+) wild-type and Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice pretreated with 5 microg dioxin (TCDD)/kg body wt or vehicle alone. NADPH-dependent H(2)O(2) production in TCDD-induced microsomes from wild-type mice was about one-third of that in noninduced microsomes. In Cyp1a2(-/-) mice, H(2)O(2) production was the same for induced and noninduced microsomes, with levels significantly higher than those in wild-type mice. Cyp1a1(-/-) microsomes displayed markedly lower levels of H(2)O(2) production in both induced and noninduced microsomes, compared with those in wild-type and Cyp1a2(-/-) microsomes. The CYP1A2 inhibitor furafylline in vitro exacerbated microsomal H(2)O(2) production proportional to the degree of CYP1A2 inhibition, and the CYP2E1 inhibitor diethyldithiocarbamate decreased H(2)O(2) production proportional to the degree of CYP2E1 inhibition. Microsomal H(2)O(2) production was strongly correlated to NADPH-stimulated production of thiobarbituric acid-reactive substances, as well as to decreases in microsomal membrane polarization anisotropy, indicative of peroxidation of unsaturated membrane lipids. Our results suggest that possibly acting as an "electron sink," CYP1A2 might decrease CYP2E1-and CYP1A1-mediated H(2)O(2) production and oxidative stress. In this regard, CYP1A2 may be considered an antioxidant enzyme.     

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.     

Resveratrol preconditioning modulates inflammatory response in the rat hippocampus following global cerebral ischemia

Considerable evidence has been accumulated to suggests that blocking the inflammatory reaction promotes neuroprotection and shows therapeutic potential for clinical treatment of ischemic brain injury. Consequently, anti-inflammatory therapies are being explored for prevention and treatment of these diseases. Induction of brain tolerance against ischemia by pretreatment with resveratrol has been found to influence expression of different molecules. It remains unclear, however, whether and how resveratrol preconditioning changes expression of inflammatory mediators after subsequent global cerebral ischemia/reperfusion (I/R). Therefore, we investigated the effect of resveratrol pretreatment on NF-κB inflammatory cascade, COX-2, iNOS and JNK levels in experimental I/R. Adult male rats were subjected to 10min of four-vessel occlusion and sacrificed at selected post-ischemic time points. Resveratrol (30mg/kg) pretreatment was injected intraperitoneally 7days prior to I/R induction. We found that resveratrol treatment before insult remarkably reduced astroglial and microglial activation at 7days after I/R. It greatly attenuated I/R-induced NF-κB and JNK activation with decreased COX-2 and iNOS production. In conclusion, the neuroprotection of resveratrol preconditioning may be due in part to the suppression of the inflammatory response via regulation of NF-κB, COX-2 and iNOS induced by I/R. JNK was also suggested to play a protective role through in neuroprotection of resveratrol, which may also be contributing to reduction in neuroinflammation. The study adds to a growing literature that resveratrol can have important anti-inflammatory actions in the brain.     

Absolute requirement for iron in the development of chemically induced uroporphyria in mice treated with 3-methylcholanthrene and 5-aminolevulinate

Accumulating evidence, including experiments using cytochrome P450 1a2 (Cyp1a2) gene knock-out mice (Cyp1a2(−/−)), indicates that the development of chemically induced porphyria requires the expression of CYP1A2. It has also been demonstrated that iron enhances and expedites the development of experimental uroporphyria, but that iron alone without CYP1A2 expression, as in Cyp1a2(−/−) mice, does not cause uroporphyria. The role of iron in the development of porphyria has not been elucidated. We examined the in vivo effect of iron deficiency on hepatic URO accumulation in experimental porphyria. Mice were fed diets containing low (iron-deficient diet (IDD), 8.5 mg iron/kg) or normal (normal diet (ND), 213.7 mg iron/kg) levels of iron. They were treated with 3-methylcholanthrene (MC), an archetypal inducer of CYP1A, and 5-aminolevulinate (ALA), precursors of porphyrin and heme. We found that uroporphyrin (URO) levels and uroporphyrinogen oxidation (UROX) activity were markedly increased in ND mice treated with MC and ALA, while the levels were not raised in IDD mice with the same treatments. CYP1A2 levels and methoxyresorufin O-demethylase (MROD) activities, the CYP1A2-mediated reaction, were markedly induced in the livers of both ND and IDD mice treated with MC and ALA. UROX activity, supposedly a CYP1A2-dependent activity, was not enhanced in iron-deficient mice in spite of the fact of induction of CYP1A2. We showed that a sufficient level of iron is essential for the development of porphyria and UROX activity.     

Silencing DJ-1 reveals its contribution in paraquat-induced autophagy

The role of autophagy as a survival strategy of cells constitutes an emerging topic in the study of the pathogenesis of several diseases with autophagic changes being described in a number of age-related neurodegenerative disorders, including Parkinson's disease (PD). Although the etiology of PD is still unknown, both environmental (for example, paraquat exposure) and genetic factors have been investigated as putative causes of the disease. In the latter case, mutations or changes in the protein DJ-1 have been reported to be associated with autosomal recessive, early-onset parkinsonism. In this paper we established a model system to study the involvement of the DJ-1 protein in paraquat-induced autophagy. When human neuroblastoma SH-SY5Y cells were transfected with DJ-1-specific small interfering RNAs and exposed to paraquat, we observed (i) sensitization additive with paraquat-induced apoptotic cell death, (ii) inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles, (iii) exacerbation of apoptotic cell death in the presence of the autophagy inhibitor 3-methyladenine, and (iv) an increase in mammalian target of rapamycin phosphorylation. Taken together, these findings suggest an active role for DJ-1 in the autophagic response produced by paraquat, providing evidence for the role of PD-related proteins in the autophagic degradation pathway, a factor that should be considered in the design of potential therapies for the treatment of the disease.     

7-Hydroperoxycholesterol as a marker of oxidative stress in rat kidney induced by paraquat

The in vivo paraquat-induced oxidative stress in rat tissue was studied by analyzing cholesterol-derived hydroperoxide as an index of lipid peroxidation. Paraquat (10 mg/kg) was administered i.p. to rats. Rats were sacrificed and lung, liver, and kidney were collected 2, 24 h, and 5 d after paraquat injection. Lipids were extracted and analyzed by HPLC with post-column chemiluminescence. We found that two cholesterol-derived hydroperoxides, 7alpha-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH) and 7beta-hydroperoxycholest-5-en-3beta-ol (7beta-OOH) were present in lungs of control animals (0.06 and 0.06 nmol/g, respectively), in livers (6.5 and 15.8 nmol/g, respectively) and in kidneys (3.7 and 8.9 nmol/g, respectively). In liver paraquat increased lipid peroxidation approximately by 60% over the levels of control animals only at 2 h after paraquat treatment. In kidney, augmented lipid peroxidation, 7alpha-OOH and 7beta-OOH (by 70% and 147%, respectively) above levels was found at 2 h after paraquat treatment. Interestingly, these increase remained in kidney of rats 5 d after a single dose of paraquat. In contrast, cholesterol-derived hydroperoxides were not affected in lung of paraquat dosed rats. This is the first report on 7alpha-OOH and 7beta-OOH accumulations in rat liver and kidney, and it seems to reflect greater oxidative stress in the pathology of kidney of rats treated with acute paraquat at low dose.     

Citicoline (CDP‐choline) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke

CDP‐choline has shown neuroprotective effects in cerebral ischemia. In humans, although a recent trial International Citicoline Trial on Acute Stroke (ICTUS) has shown that global recovery is similar in CDP‐choline and placebo groups, CDP‐choline was shown to be more beneficial in some patients, such as those with moderate stroke severity and not treated with t‐PA. Several mechanisms have been proposed to explain the beneficial actions of CDP‐choline. We have now studied the participation of Sirtuin1 (SIRT1) in the neuroprotective actions of CDP‐choline. Fischer rats and Sirt1^?/? mice were subjected to permanent focal ischemia. CDP‐choline (0.2 or 2 g/kg), sirtinol (a SIRT1 inhibitor; 10 mg/kg), and resveratrol (a SIRT1 activator; 2.5 mg/kg) were administered intraperitoneally. Brains were removed 24 and 48 h after ischemia for western blot analysis and infarct volume determination. Treatment with CDP‐choline increased SIRT1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP‐choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP‐choline. CDP‐choline failed to reduce infarct volume in Sirt1^?/? mice. Our present results demonstrate a robust effect of CDP‐choline like SIRT1 activator by up‐regulating its expression. Our findings suggest that therapeutic strategies to activate SIRT1 may be useful in the treatment of stroke.

     

Rifampicin-induced CYP3A4 activation in CTX patients cannot replace chenodeoxycholic acid treatment

Cerebrotendinous xanthomatosis (CTX) is a rare neurodegenerative disorder with cholestanol accumulation resulting from mutations in the sterol 27-hydroxylase gene (CYP27A). Conventional treatment includes chenodeoxycholic acid and HMG-CoA reductase inhibitors. Mice with disrupted Cyp27A (Cyp27 KO) do not show elevated cholestanol levels nor develop CTX manifestations. This phenomenon was proposed to be due to murine CYP3A overexpression leading to an alternative pathway for degradation of bile alcohols including cholestanol. Our objective was to examine the influence of CYP3A4 induction on cholestanol elimination in CTX patients. Rifampicin (600 mg/day x 7 days), known to induce the PXR, and thereby to increase CYP3A activity, was used. The degree of CYP3A4 induction was assessed by comparing midazolam pharmacokinetics before and after rifampicin treatment. Cholestanol levels and cholestanol/cholesterol ratios were assayed during the experimental period and compared to a 3 weeks period without treatment. The results show that despite 60% increase in CYP3A4 activity following rifampicin treatment, there is no significant change in cholestanol levels. We conclude that up-regulated expression of CYP3A affects cholestanol elimination in mice differently as compared to its effect in CTX patients. Therefore, CYP3A4 inducers cannot replace chenodeoxycholic acid for the treatment of CTX.     

Effects of L-dopa and other amino acids against paraquat-induced nigrostriatal degeneration

Exposure to the herbicide paraquat causes selective nigrostriatal degeneration and aggregation of alpha-synuclein in the mouse brain. The purpose of this study was to assess mechanisms of paraquat entry into the CNS and, in particular, the effects of substrates of the blood-brain barrier (BBB) neutral amino acid transporter (System L carrier) on paraquat accumulation and neurotoxicity. Using a paraquat antibody, robust immunoreactivity was observed in the midbrain of mice injected with the herbicide. This immunoreactivity was abolished by administration of l-valine or l-phenylalanine, two System L substrates, immediately before paraquat exposure. Pre-treatment with these amino acids completely protected against paraquat-induced loss of nigrostriatal dopaminergic cells and formation of thioflavine S-positive intracellular deposits. Interestingly, the anti-parkinsonian drug l-dopa, which is transported across the BBB through the same neutral amino acid carrier, was also neuroprotective when administered 30 min prior to paraquat. In contrast, paraquat-induced toxicity was unaffected if animals (i) were pre-treated with d-valine, the biologically inactive d-isomer of l-valine, or with l-lysine, a substrate of the basic rather than the neutral amino acid carrier, or (ii) were injected with l-dopa 24 h after paraquat exposure. Data are consistent with a critical role of uptake across the BBB in paraquat neurotoxicity, and suggest that dietary elements (e.g. amino acids) or therapeutic agents (e.g. l-dopa) may modify the effects of toxicants targeting the nigrostriatal system.     

Selective estrogen receptor modulators protect hippocampal neurons from kainic acid excitotoxicity: differences with the effect of estradiol

Neuroprotective effects of estradiol are well characterized in animal experimental models. However, in humans, the outcome of estrogen treatment for cognitive function and neurological diseases is very controversial. Selective estrogen receptor modulators (SERMs) may represent an alternative to estrogen for the treatment or the prevention of neurodegenerative disorders. SERMs interact with the estrogen receptors and have tissue-specific effects distinct from those of estradiol, acting as estrogen agonists in some tissues and as antagonists in others. In this study we have assessed the effect of tamoxifen, raloxifene, lasofoxifene (CP-336,156), bazedoxifene (TSE-424), and 17beta-estradiol on the hippocampus of adult ovariectomized rats, after the administration of the excitotoxin kainic acid. Administration of kainic acid induced the expression of vimentin in reactive astroglia and a significant neuronal loss in the hilus. SERMs did not affect vimentin immunoreactivity in the hilus, while 17beta-estradiol significantly reduced the surface density of vimentin immunoreactive profiles. Estradiol, tamoxifen (0.4-2 mg/kg), raloxifene (0.4-2 mg/kg), and bazedoxifene (2 mg/kg) prevented neuronal loss in the hilus after the administration of kainic acid. Lasofoxifene (0.4-2 mg/kg) was not neuroprotective. These findings indicate that SERMs present different dose-dependent neuroprotective effects. Furthermore, the mechanisms of neuroprotection by SERMs and estradiol are not identical, because SERMs do not significantly affect reactive gliosis while neuroprotection by estradiol is associated with a strong down-regulation of reactive astroglia.     

Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.     

Relative roles of CYP2E1 and CYP1A2 in mouse uroporphyria caused by acetone

Porphyria cutanea tarda is a liver disease characterized by excess production of uroporphyrin. We previously reported that acetone, an inducer of CYP2E1, enhances hepatic uroporphyrin accumulation in mice treated with iron dextran (Fe) and 5-aminolevulinic acid (ALA). Cyp2e1(-/-) mice treated with Fe and ALA were used to investigate whether CYP2E1 is required for the acetone effect. Hepatic uroporphyrin accumulation was stimulated by acetone in Cyp2e1(-/-) mice to the same extent as in wild-type mice. In the absence of acetone, uroporphyrin accumulated in Cyp2e1(-/-) mice treated with Fe and ALA, but less than in wildtype mice. However, in Cypla2(-/-) mice, uroporphyrin accumulation caused by Fe and ALA, with or without acetone, was completely prevented. Acetone was not an inducer of hepatic CYP1A2 in the wild-type mice. Although acetone is an inducer of CYP2E1, CYP1A2 appears to have the essential role in acetone-enhancement of uroporphyria.     

Selenium-dependent cellular glutathione peroxidase protects mice against a pro-oxidant-induced oxidation of NADPH, NADH, lipids, and protein.

Since our prior work indicated that Se-dependent cellular glutathione peroxidase (GPX1) was necessary for protection against paraquat lethality, the present studies were to elucidate the biochemical mechanisms related to that protection. Four groups of mice [Se-deficient or -adequate GPX1 knockout and wild-type (WT)] were injected (i.p.) with 50 mg paraquat/kg body weight and tissues were collected 0, 0.5, 1, 2, 3, or 4 h after the injection. Whereas the ratios of NADPH/NADP and NADH/NAD in lung were reduced by 50-70% only 0.5 h after the injection in all groups, these two ratios in liver of the Se-adequate WT were significantly higher than those of the three GPX1 knockout or deficient groups 2-4 h after the injection. The paraquat-induced pulmonary lipid peroxidation and hepatic protein oxidation, measured as F(2)-isoprostanes and carbonyl contents, respectively, peaked at 1 h in these three groups. No such oxidative events were shown in any tissue of the Se-adequate WT throughout the time course. Whereas the F(2)-isoprostane formation was accelerated by both GPX1 knockout and Se deficiency in liver, it was not significantly elevated by the paraquat treatment in brain of any group. The paraquat injection also resulted in temporal changes in lung GPX activity and GPX1 protein in the Se-adequate WT, and significant reductions in lung total SOD activity in the GPX1 knockout or deficient groups. In conclusion, GPX1 plays a critical role in maintaining the redox status of mice under acute oxidative stress, and protects against paraquat-induced oxidative destruction of lipids and protein in vivo. These protections of GPX1 seem to be inducible and coordinated with those of other antioxidant enzymes.     

Mitochondrial reactive oxygen production is dependent on the aromatic hydrocarbon receptor

2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is a pervasive environmental contaminant that induces hepatic and extrahepatic oxidative stress. We have previously shown that dioxin increases mitochondrial respiration-dependent reactive oxygen production. In the present study we examined the dependence of mitochondrial reactive oxygen production on the aromatic hydrocarbon receptor (AHR), cytochrome P450 1A1 (CYP1A1), and cytochrome P450 1A2 (CYP1A2), proteins believed to be important in dioxin-induced liver toxicity. Congenic Ahr(-/-), Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice, and C57BL/6J inbred mice as their Ahr/Cyp1a1/Cyp1a2(+/+) wild-type (wt) counterparts, were injected intraperitoneally with dioxin (15 microg/kg body weight) or corn-oil vehicle on 3 consecutive days. Liver mitochondria were examined 1 week following the first treatment. The level of mitochondrial H(2)O(2) production in vehicle-treated Ahr(-/-) mice was one fifth that found in vehicle-treated wt mice. Whereas dioxin caused a rise in succinate-stimulated mitochondrial H(2)O(2) production in the wt, Cyp1a1(-/-), and Cyp1a2(-/-) mice, this increase did not occur with the Ahr(-/-) knockout. The lack of H(2)O(2) production in Ahr(-/-) mice was not due to low levels of Mn(2+)-superoxide dismutase (SOD2) as shown by Western immunoblot analysis, nor was it due to high levels of mitochondrial glutathione peroxidase (GPX1) activity. Dioxin decreased mitochondrial aconitase (an enzyme inactivated by superoxide) by 44% in wt mice, by 26% in Cyp1a2(-/-) mice, and by 24% in Cyp1a1(-/-) mice; no change was observed in Ahr(-/-) mice. Dioxin treatment increased mitochondrial glutathione levels in the wt, Cyp1a1(-/-), and Cyp1a2(-/-) mice, but not in Ahr(-/-) mice. These results suggest that both constitutive and dioxin-induced mitochondrial reactive oxygen production is associated with a function of the AHR, and these effects are independent of either CYP1A1 or CYP1A2.