Biomedical subjects

Howard G Shertzer

Publications and source records attributed to Howard G Shertzer.

8 recordsLinked to original sources

PubMed2003

4-aminobiphenyl-induced liver and urinary bladder DNA adduct formation in Cyp1a2(-/-) and Cyp1a2(+/+) mice.

BACKGROUND: Metabolites of the potent human carcinogen 4-aminobiphenyl (ABP) induce oxidative stress and form DNA adducts that are associated with hepatic and urinary bladder toxicity and bladder tumorigenesis. Results of in vitro and cell culture studies have suggested that cytochrome P450 1A2 (CYP1A2) is the major metabolic activator of ABP. We used Cyp1a2(-/-) knockout mice to examine the role of CYP1A2 in ABP-DNA adduct formation in the liver and the bladder. METHODS: Cyp1a2(+/+) wild-type and Cyp1a2(-/-) mice (total of four mice per group) were treated topically with 10 mg/kg ABP for various times, with or without pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an inducer of CYP1A2 activity. We evaluated ABP-induced toxicity by carrying out quantitative histology (of the liver, skin, and bladder), oxidative stress by measuring hepatic thiol levels, and liver and bladder DNA adduct formation by using 32P-postlabeling. Data were analyzed by general linear models and analysis of variance. All statistical tests were two-sided. RESULTS: At the experimental times selected, we observed no histologic evidence of toxicity in the liver, skin, or bladder. Overall, Cyp1a2(+/+) mice had fewer DNA adducts 24 hours after ABP treatment than similarly treated Cyp1a2(-/-) mice. Compared with male mice, female mice had more DNA adducts in the liver but fewer adducts in the bladder, regardless of Cyp1a2 genotype. TCDD pretreatment was associated with a decrease in ABP-DNA adduct levels overall. After 2 hours of ABP treatment, hepatic thiol levels underwent statistically significant declines of severalfold in Cyp1a2(+/+) and Cyp1a2(-/-) males and in Cyp1a2(-/-) females. CONCLUSIONS: Contrary to our expectations, CYP1A2 expression was not associated with ABP-induced hepatic oxidative stress or with ABP-DNA adduct formation. Either CYP1A2 is not the major metabolic activator of ABP or other enzymes metabolically activate ABP in mice in the absence of CYP1A2.

Aminobiphenyl Compounds↗

PubMed2003

Pharmacological rescue of the 14CoS/14CoS mouse: hepatocyte apoptosis is likely caused by endogenous oxidative stress.

Whereas ch/ch wild-type mice and ch/14CoS heterozygotes are viable, 14CoS/14CoS mice homozygous for a 3800 kb deletion on chromosome 7 die during the first day postpartum. Death is caused by disruption of the fumarylacetoacetate hydrolase (Fah) gene; absence of FAH, final enzyme in the tyrosine catabolism pathway, leads to accumulation of reactive electrophilic intermediates. In this study, we kept 14CoS/14CoS mice alive for 60 d with oral 2-(2-nitro-4-trifluoromethyl-benzyol)-1,3-cyclohexanedione (NTBC), an inhibitor of p-hydroxyphenylpyruvate dioxygenase, second enzyme in the tyrosine catabolic pathway. The 70% of NTBC-treated 14CoS/14CoS mice that survived 60 d showed poor growth and developed corneal opacities, compared with ch/14CoS littermates; NTBC-rescued Fah(-/-) knockout mice did not show growth retardation or ocular toxicity. NTBC-rescued 14CoS/14CoS mice also exhibited a striking oxidative stress response in liver and kidney, as measured by lower GSH levels and mRNA induction of four genes: glutamate cysteine ligase catalytic (Gclc) and modifier (Gclm) subunits, NAD(P)H:quinone oxidoreductase (Nqo1), and heme oxygenase-1 (Hmox1). Withdrawal of NTBC for 24-48 h from rescued adult 14CoS/14CoS mice resulted in severe apoptosis of the liver, detected histologically and by cytochrome c release from the mitochondria, increased caspase 3-like activity, and further decreases in GSH content. In kidney, proximal tubular epithelial cells were abnormal. Human hereditary tyrosinemia type I (HT1), caused by mutations in the FAH gene, is an autosomal recessive disorder in which the patient usually dies of liver fibrosis and cirrhosis during early childhood; NTBC treatment is known to prolong HT1 children's lives-although liver fibrosis, cirrhosis, hepatocarcinoma, and corneal opacities sometimes occur. The mouse data in the present study are consistent with the possibility that endogenous oxidative stress-induced apoptosis may be the underlying cause of liver pathology seen in NTBC-treated HT1 patients.

Animals↗

PubMed2002

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.

Aconitate Hydratase↗

PubMed2002

Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response.

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(-/-) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(-/-) mice were 9-16% of that in Gclm(+/+) littermates. Cysteine levels in Gclm(-/-) mice were 9, 35, and 40% of that in Gclm(+/+) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an approximately 2-fold increase in K(m) for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(-/-) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H(2)O(2). We conclude that the Gclm(-/-) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.

Age Factors↗

PubMed2002

Induction of cellular oxidative stress by aryl hydrocarbon receptor activation.

The aryl hydrocarbon receptor (AHR) has long been associated with the induction of a battery of genes involved in the metabolism of foreign and endogenous compounds. Depending on experimental conditions, AHR can mediate either activation or amelioration of chemical toxicity. For the past decade, evidence has mounted that AHR is associated with a cellular oxidative stress response that must be considered when evaluating the mechanism of action of xenobiotics capable of activating AHR, or capable of metabolic activation by enzymes encoded by genes under control of AHR. In this review, we have evaluated the diverse mechanisms by which AHR generates an oxidative stress response, including inflammation, antioxidant and prooxidant enzymes and cytochrome P450. A review of the regulation of Ahr transcription and functional polymorphisms especially related to oxidative stress is also included. We have carefully avoided placing a value judgment on the degree of toxicity produced by such a response, in view of the realization that an oxidative response is involved in many normal physiological processes. Since the interface between physiological, adaptive and toxicological responses elicited by the AHR-mediated oxidative stress response is not clearly defined, it behooves the researcher to evaluate both toxicological and physiological features of the response.

Animals↗

PubMed2002

Decrease in 4-aminobiphenyl-induced methemoglobinemia in Cyp1a2(-/-) knockout mice.

Methemoglobin formation, as well as hemoglobin or DNA adducts, are useful biomarkers of occupational exposure to certain arylamines. It has been suggested that, in liver from animals not treated with a cytochrome P450 (CYP) inducer, hepatic CYP1A2 is the major P450 involved in N-hydroxylation. This is the first step in the metabolic activation of many arylamines, such as the human urinary bladder carcinogen 4-aminobiphenyl (ABP). The product of this catalytic step, N-hydroxy-4-ABP, reacts in the blood with oxyhemoglobin to form methemoglobin and nitrosobiphenyl. We therefore examined the role of CYP1A2 in causing methemoglobinemia in ABP-treated Cyp1a2(-/-) knockout mice. Application of ABP (100 micromol/kg body wt) to the skin resulted in a marked depletion in the levels of the hepatic thiols (reduced glutathione and cysteine) after 2 h, which rebounded to basal levels 24 h later, and we found no differences between the Cyp1a2(-/-) and wild-type Cyp1a2(+/+) animals. Unexpectedly, the methemoglobin levels were significantly (p < 0.05) higher in Cyp1a2(-/-) than Cyp1a2(+/+) mice at 2, 7, and 24 h following topical ABP. Treatment with dioxin, 24 h prior to ABP, decreased methemoglobin levels by about half at each of the time points in both the Cyp1a2(-/-) and Cyp1a2(+/+) mice. These data suggest that CYP1A2 does not play a positive role in methemoglobin formation via the activation of ABP; rather, the absence of CYP1A2 enhances ABP-induced methemoglobinemia. Because liver CYP1A2 levels are known to vary more than 60-fold between humans, our findings may be relevant to patients who are exposed to arylamines in the workplace.

Administration, Topical↗

PubMed2002

Glutamate-cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress.

Glutamate-cysteine ligase is a heterodimer comprising a modifier (GCLM) and a catalytic (GCLC) subunit. In mouse Hepa-1c1c7 hepatoma cell cultures, we found that tert-butylhydroquinone (tBHQ; 50 microM) induces the GCLM and GCLC mRNAs approximately 10- and approximately 2-fold, respectively, and that these increases primarily reflect de novo transcription. We determined that the mouse Gclm gene has seven exons, spanning 22.3 kb; all exons, intron-exon junctions, and 4.7 kb of 5'-flanking region were sequenced. By RNase protection analysis, we identified two major and several minor transcription start-site clusters over a 300-bp region. The Gclm 5'-flanking region is GC-rich and lacks a canonical TATA box. Transient and stable transfection studies, using luciferase reporter constructs containing incremental Gclm 5'-flanking deletions (4.7-0.5 kb), showed high basal activity but only modest ( approximately 2-fold) inducibility by tBHQ. The only candidate motif for oxidative stress regulation (in the 4.7-kb region we sequenced) is a putative inverted electrophile response element (EPRE) 9 bp upstream from the 5'-most transcription start-site. Site-directed mutagenesis of this -9 EPRE demonstrated minimal (30-40%) decreases in tBHQ induction and no effect on basal activity-suggesting that this EPRE might be necessary but not sufficient. The nuclear erythroid factor-2 (NEF2)-related factor-2 (NRF2) is known to transactivate via EPRE motifs. In the presence of co-transfected NRF cDNA expression vector, however, no increase in Gclm promoter activity was observed. Thus, the endogenous Gclm gene shows robust transcriptional activation by tBHQ in the intact Hepa-1 cell, but reporter constructs containing up to 4.7 kb of promoter (having only the one EPRE at -9) demonstrate a disappointing response, indicating that the major tBHQ-responsive regulatory element of the mouse Gclm gene must exist either further 5'- or 3'-ward of the 4.7-kb region studied.

5' Flanking Region↗

PubMed2002

Dioxin increases reactive oxygen production in mouse liver mitochondria.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) causes an oxidative stress response in liver and several extrahepatic tissues. The subcellular sources and underlying mechanisms of dioxin-induced reactive oxygen, however, are not well understood. In this study, we examined whether mitochondria, organelles that consume the majority of cellular oxygen, might be a source of dioxin-induced reactive oxygen. Female C57BL/6 mice were treated with dioxin (15 microg/kg body wt ip) on 3 consecutive days, and liver mitochondria were examined at 1, 4, and 8 weeks after the first treatment. Mitochondrial aconitase activity, an enzyme inactivated by superoxide, was decreased by 44% at 1 week, 22% at 4 weeks, and returned to control levels at 8 weeks. Dioxin elevated succinate-stimulated mitochondrial H2O2 production twofold at 1 and 4 weeks; H2O2 production remained significantly elevated at 8 weeks. The enhanced H2O2 production was due to neither increased Mn-superoxide dismutase activity nor decreased mitochondrial glutathione peroxidase activity. Dioxin treatment augmented mitochondrial glutathione, but not glutathione disulfide levels, a result that might be explained by increased mitochondrial glutathione reductase activity. Liver ATP levels were significantly lowered at 1 and 4 weeks, the peak times of mitochondrial reactive oxygen production. Increased dioxin-stimulated reactive oxygen at 1 and 4 weeks did not appear to be related to the observed decrease in cytochrome oxidase activity, since State 3 and State 4 respiration were not diminished. To our knowledge, this is the first report to show that dioxin increases mitochondrial respiration-dependent reactive oxygen production, which may play an important role in dioxin-induced toxicity and disease.

Aconitate Hydratase↗