Identification of Environmental Chemicals Associated with the Development of Toxicant-associated Fatty Liver Disease in Rodents.

BACKGROUND: Toxicant-associated fatty liver disease (TAFLD) is a recently identified form of nonalcoholic fatty liver disease (NAFLD) associated with exposure to industrial chemicals and environmental pollutants. Numerous studies have been conducted to test the association between industrial chemicals/environmental pollutants and fatty liver disease both in vivo and in vitro. OBJECTIVES: The objective of the article is to report a list of chemicals associated with TAFLD. METHODS: Two federal databases of rodent toxicology studies-Toxicological Reference Database (ToxRefDB; Environmental Protection Agency) and Chemical Effects in Biological Systems (CEBS, National Toxicology Program)-were searched for liver end points. Combined, these 2 databases archive nearly 2,000 rodent studies. Toxicant-associated steatohepatitis (TASH) descriptors including fatty change, fatty necrosis, Oil red O-positive staining, steatosis, and lipid deposition were queried. RESULTS: Using these search terms, 123 chemicals associated with fatty liver were identified. Pesticides and solvents were the most frequently identified chemicals, while polychlorinated biphenyls (PCBs)/dioxins were the most potent. About 44% of identified compounds were pesticides or their intermediates, and >10% of pesticide registration studies in ToxRefDB were associated with fatty liver. Fungicides and herbicides were more frequently associated with fatty liver than insecticides. CONCLUSION: More research on pesticides, solvents, metals, and PCBs/dioxins in NAFLD/TAFLD is warranted due to their association with liver damage.

Activator protein-1 regulation of murine aldehyde dehydrogenase 1a1.

Previously we demonstrated that aldehyde dehydrogenase (ALDH) 1a1 is the major ALDH expressed in mouse liver and is an effective catalyst in metabolism of lipid aldehydes. Quantitative real-time polymerase chain reaction analysis revealed a ≈2.5- to 3-fold induction of the hepatic ALDH1A1 mRNA in mice administered either acrolein (5 mg/kg acrolein p.o.) or butylated hydroxylanisole (BHA) (0.45% in the diet) and of cytosolic NAD⁺-dependent ALDH activity. We observed ≈2-fold increases in ALDH1A1 mRNA levels in both Nrf2⁺/⁺ and Nrf2⁻/⁻ mice treated with BHA compared with controls, suggesting that BHA-induced expression is independent of nuclear factor E2-related factor 2 (Nrf2). The levels of activator protein-1 (AP-1) mRNA and protein, as well as the amount of phosphorylated c-Jun were significantly increased in mouse liver or Hepa1c1c7 cells treated with either BHA or acrolein. With use of luciferase reporters containing the 5'-flanking sequence of Aldh1a1 (-1963/+27), overexpression of c-Jun resulted in an ≈4-fold induction in luciferase activity, suggesting that c-Jun transactivates the Aldh1a1 promoter as a homodimer and not as a c-Jun/c-Fos heterodimer. Promoter deletion and mutagenesis analyses demonstrated that the AP-1 site at position -758 and possibly -1069 relative to the transcription start site was responsible for c-Jun-mediated transactivation. Electrophoretic mobility shift assay analysis with antibodies against c-Jun and c-Fos showed that c-Jun binds to the proximal AP-1 site at position -758 but not at -1069. Recruitment of c-Jun to this proximal AP-1 site by BHA was confirmed by chromatin immunoprecipitation analysis, indicating that recruitment of c-Jun to the mouse Aldh1a1 gene promoter results in increased transcription. This mode of regulation of an ALDH has not been described before.

Regulation of the rat UGT1A6 by glucocorticoids involves a cryptic glucocorticoid response element.

Glucocorticoids precociously induce fetal rat UGT1A6 and potentiate polycyclic aromatic hydrocarbon (PAH)-dependent induction of this enzyme in vivo and in isolated rat hepatocytes. To establish whether induction was due to glucocorticoid receptor (GR), luciferase reporter vectors were tested in transfection assays with HepG2 cells. Using a reporter construct containing approximately 2.26 kilobases of the 5'-flanking region of the UGT1A6-noncoding leader exon (A1*), dexamethasone increased basal activity 3- to 7-fold in cells cotransfected with an expression plasmid for GR. PAH increased gene expression 23-fold, but the presence of dexamethasone only induced PAH-dependent expression by 1.5-fold, suggesting interaction between GR and the aryl hydrocarbon (Ah) receptor. Furthermore, the GR antagonist RU 38486 [17beta-hydroxy-11beta-(4-dimethylamino-phenyl)-17alpha-(prop-1-ynyl)-estra-4,9-dien-3-one] was a partial agonist that increased, rather than inhibited, basal activity 3-fold. 5'-deletion analysis defined the 5'-boundary for a functional glucocorticoid-responsive unit between base pairs -141 and -118 relative to the transcription start site. This region contains the Ah receptor response element (AhRE), and both PAH and glucocorticoid-dependent gene activation were lost when this area was deleted. Mutation of a single base pair located in the AhRE region simultaneously reduced induction by PAH and increased glucocorticoid induction. Thus, the sequences of both the AhRE and glucocorticoid response elements seem to overlap, suggesting that Ah receptor binding may decrease glucocorticoid-dependent induction due to interactions of these two cis-acting elements. Mutation of a putative GRE located between base pair -81 and -95 reduced, but did not completely eliminate, glucocorticoid-dependent induction of the reporter, suggesting that a nonclassic mechanism of induction is involved in this response.

Regulation of NAD(P)H:quininone oxidoreductase by glucocorticoids.

Previous studies in neonatal and adolescent rats as well as adrenalectomized rats have demonstrated that glucocorticoids regulate the expression of the rat NAD(P)H:quinone oxidoreductase gene (QOR). We used primary cultures of rat adult hepatocytes to document that added glucorticoids repress both the basal and 1,2-benzanthracene-induced expression of QOR mRNA by 65-70%. QOR enzyme activity and protein were concomitantly suppressed as well. The monotonic concentration response for repression of QOR gene products up to 100 microM DEX concentration demonstrated that the glucocorticoid receptor (GR) was most likely involved in this process. The lack of effect at higher concentration rules out a role for the Pregnane X receptor in this regulation by DEX. In addition, the anti-glucorticoid RU38486 blocked this negative regulation and the protein synthesis inhibitor cycloheximide had no effect on this repression process. Similar results of GR dependence were observed using a luciferase reporter construct containing the 5'-flanking region of the human QOR gene using HepG2 cells. Collectively, these results demonstrate that GR must directly participate in the negative regulation of QOR gene expression by dexamethasone and other glucocorticoids in vivo.

Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: involvement of both the glucocorticoid and pregnane X receptors.

Glucocorticoids regulate the rat glutathione S-transferase A2 (GSTA2) gene in a biphasic manner in cultured hepatocytes that repress gene expression at low concentration (10--100 nM) but induce gene expression at high concentration (>1 microM). High concentrations of the glucocorticoid receptor (GR) antagonist RU38486 (5--10 microM) also induced the expression of GSTA2. These effects were reproduced in HepG2 cells transfected with a luciferase reporter containing 1.6 kilobase pairs of 5'-flanking sequence of GSTA2 and expression plasmids for either GR, pregnane X receptor (PXR) or a combination of both. Dexamethasone t-butylacetate (1 microM t-Bu-DEX) repressed gene expression between 60 to 75% when only GR was expressed. When PXR was expressed, both basal and t-Bu-DEX-dependent gene expression was increased over 2-fold, respectively. Biphasic regulation of gene expression was observed over a broad range of t-Bu-DEX concentrations when expression plasmids for both receptors were cotransfected. Other steroids of the pregnane class induced GSTA2 expression as expected for a PXR-dependent process. Because no canonical responsive element for the PXR-RXR alpha heterodimer was observed in the 5'-flanking region of the construct, deletion analysis was used to identify a pregnane responsive region between base pairs -700 and -683; this 20-bp region contains the antioxidant response element (ARE). When the ARE sequence was mutated, basal, t-butylhydroquinone- and 17 alpha-hydroxypregnenolone-inducible expression were all lost. These results suggest that PXR interacts with factors binding to the ARE to elicit the pregnane inductive response for GSTA2.

Short heterodimer partner (SHP) orphan nuclear receptor inhibits the transcriptional activity of aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT).

SHP (short heterodimer partner) is an orphan nuclear receptor lacking a DNA binding domain that interacts with nuclear receptors (NR) including thyroid receptor (TR), retinoic acid receptors (RAR and RXR), and estrogen receptors alpha and beta (ERalpha and ERbeta). SHP acts as a negative regulator of these receptors by inhibiting DNA binding and transcriptional activation. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to arylhydrocarbon receptor (AHR), activating the AHR/AHR nuclear translocator (ARNT) heterodimer. We investigated the physical and functional interaction of SHP with AHR/ARNT. In RL95-2 human endometrial carcinoma cells, SHP inhibited TCDD-stimulated reporter activity from the AHR-responsive CYP1A1 and UGT1A6 gene promoters in a concentration-dependent manner. In GST pull-down assays, ARNT interacted directly with SHP in vitro, but AHR did not interact with GST-SHP. SHP inhibited AHR/ARNT-DNA binding in vitro. These results identify ARNT as a novel SHP target. We speculate a role for SHP in the suppression of agonist-activated AHR/ARNT activity.

Molecular regulation of genes encoding xenobiotic-metabolizing enzymes: mechanisms involving endogenous factors.

It is widely recognized that xenobiotic-metabolizing enzymes play a fundamental role in the basic processes of carcinogenesis and toxicity on one hand, and chemoprevention and drug efficacy on the other. Realization that different factors can profoundly affect the expression of these enzymes at the genome level has resulted in an enhanced appreciation of the importance these genes play in our modern industrialized age. There continues to be rapid proliferation of studies addressing the molecular regulation of these genes. The discovery of common signal transduction pathways and transcription factors that dictate tissue and developmental-specific expression, as well as variation in expression within a given tissue, suggest that there may be significant interaction among these various regulatory systems. This report is a summary of a symposium that was part of the Structure, Function and Regulation of Cytochromes P450 and Xenobiotic Metabolizing Enzymes satellite meeting of the 2000 joint meeting of the American Society for Biochemistry and Molecular Biology, the American Society for Pharmacology and Experimental Therapeutics, the French Pharmacological Society, and the Pharmacological Society of Canada held in Boston, Massachusetts. This symposium brought together several speakers who addressed specific receptor-mediated signal transduction pathways involved in the regulation of xenobiotic-metabolizing enzymes, as well as other molecular mechanisms whereby endogenous factors are involved in controlling tissue- and developmental-specific expression.

Negative regulation of rat hepatic aldehyde dehydrogenase 3 by glucocorticoids.

The expression of the aldehyde dehydrogenase 3 gene is known to be controlled by multiple regulatory processes. In liver, inducible expression appears to be mediated by two AhRE sequences which allow regulation of this gene by xenobiotic compounds which are ligands for the Ah receptor (Takimoto et al., 1994; this work). Constitutive expression of ALDH3 in tissues such as the cornea also involves the -3,500 region which contains an AhRE (Boesch et al., 1996; Boesch et al, 1998). However, the constellation of transcription factors which appear to interact with the AhRE in constitutively expressing corneal cells does not include either the Ah receptor nor the prototypical ARNT protein (Boesch et al., 1998). For both inducible and constitutive ALDH3 expression the more distal 5' flanking region sequences appear to interact with more proximal regulatory elements. Of particular interest is the region near -1 kb which includes the GC (-930 to -910) and cAMP (-1057 to -991) responsive elements as well as the 2 NF1 sites (-916 to -815), all of which appear to act as negative modulators of ALDH3 expression. A second putative ALDH3 negative regulatory region lies even more distal than -3,500 bp. To date, this region has been little studied, but appears to be involved in regulating both inducible and constitutive ALDH3 expression. This region may also be responsible for some of the tissue-specificity of ALDH3 expression. With respect to the work described here, in both isolated hepatocytes and HepG2 cells, no consistent negative regulation by glucocorticoids was observed in the basal expression of ALDH3. This indicates that the mechanism of GC-mediated negative regulation involves direct interference with ALDH3 gene activation mediated by the Ah receptor. Our results suggest a complex interplay between multiple transcription factors, including the GC and Ah receptors, regulates the hepatic expression of the ALDH3 gene. Active recruitment of transcription factors needed for gene transactivation, amelioration of the actions of negative regulatory trans-acting factors or cis-acting elements and/or chromatin remodeling may be required for achieve proper regulation of the aldehyde dehydrogenase 3 gene.

The negative regulation of the rat aldehyde dehydrogenase 3 gene by glucocorticoids: involvement of a single imperfect palindromic glucocorticoid responsive element.

Glucocorticoids repressed the polycyclic aromatic hydrocarbon-dependent induction of Class 3 aldehyde dehydrogenase (ALDH3) enzyme activity and mRNA levels in isolated rat hepatocytes by more than 50 to 80%, with a concentration-dependence consistent with the involvement of the glucocorticoid receptor (GR). No consistent effect on the low basal transcription rate was observed. This effect of glucocorticoids (GC) on polycyclic aromatic hydrocarbon induction was effectively antagonized at the mRNA and protein level by the GR antagonist RU38486. The response was cycloheximide-sensitive, because the protein synthesis inhibitor caused a GC-dependent superinduction of ALDH3 mRNA levels. This suggests that the effects of GC on this gene are complex and both positive and negative gene regulation is possible. The GC-response was recapitulated in HepG2 cells using transient transfection experiments with CAT reporter constructs containing 3.5 kb of 5'-flanking region from ALDH3. This ligand-dependent response was also observed when a chimeric GR (GR DNA-binding domain and peroxisome proliferator-activated receptor ligand-binding domain) was used in place of GR in the presence of the peroxisome proliferator, nafenopin. A putative palindromic glucocorticoid-responsive element exists between -930 and -910 base pairs relative to the transcription start site. If this element was either deleted or mutated, the negative GC-response was completely lost, which suggests that this sequence is responsible, in part, for the negative regulation of the gene. Electrophoretic mobility shift analysis demonstrated that this palindromic glucocorticoid-responsive element is capable of forming a specific DNA-protein complex with human glucocorticoid receptor. In conclusion, the negative regulation of ALDH3 in rat liver is probably mediated through direct GR binding to its canonical responsive element.

Negative regulation of the rat glutathione S-transferase A2 gene by glucocorticoids involves a canonical glucocorticoid consensus sequence.

Glucocorticoids (GCs) repress both basal and polyaromatic hydrocarbon-induced expression of the glutathione S-transferase Ya1 gene (gstA2) in isolated rat hepatocytes and rat liver in vivo. Transient transfection experiments with HepG2 cells were used to identify GC-responsive elements (GREs). With cotransfected GC receptor, chloramphenicol acetyltransferase (CAT) constructs containing a palindromic GRE (pGRE) and three GRE hexanucleotide half-sites between -1.6 and -1.1 kb of the 5'-flanking region of gstA2 were repressed >50% by GC when induced with polyaromatic hydrocarbon. This pGRE, if either mutated or deleted, significantly reduces GC responsiveness of the gene to 20-30%; no effect of GC was observed with CAT constructs containing -1.15 kb of the 5'-flanking region. The dexamethasone concentration dependence of the repression was consistent with involvement of the GC receptor and was antagonized by RU38486. Electrophoretic mobility shift assays demonstrated that pGRE formed a specific DNA/protein complex, which was prevented by the addition of excess unlabeled or mouse mammary tumor virus GRE but not by unrelated or mutated gstA2 GRE double-stranded oligonucleotides. This complex was supershifted by incubation of nuclear extracts containing GC receptor with anti-GC receptor globulins. Constructs containing multiple copies of pGRE sequence were either nonresponsive or positively responsive (three copies) to GC. Luciferase constructs containing -1.62 to -1.03 kb of the 5'-flanking region also were regulated positively by GC. Chimeric GC-peroxisome proliferator activated receptor activated the constructs that were positively responsive to GC but did not mediate the negative effect in constructs containing 1.6 kb of 5'-flanking region. We conclude that pGRE and half-site GREs of gstA2 participate in regulation of this gene; however, a second unidentified responsive element must exist between -1.03 and -0.164 kb, resulting in repression of gstA2 expression.

cAMP-dependent negative regulation of rat aldehyde dehydrogenase class 3 gene expression.

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.

Hormonal regulation of hepatic enzymes involved in foreign compound metabolism.

The regulation of hepatic P450s has been the focus of numerous studies because of the importance of these proteins in endocrinology, oncology, and toxicology, as well as drug development. Considerable evidence exists demonstrating that many hepatic P450s are regulated by developmental, sex, or hormonal factors in addition to receptors that interact with foreign chemicals. The focus of work in our laboratory has been on the effects of steroid hormones, especially glucocorticoids, on expression of genes regulated by the Ah receptor. We have shown that most rat hepatic genes of the Ah receptor gene battery are regulated by glucocorticoids. We have used glucocorticoid-deficient animal models to demonstrate that these steroids do modulate the expression (basal and inducible) of these genes in vivo. Using cultured rat hepatocytes, we have demonstrated that polycyclic aromatic hydrocarbon (PAH) induction of cytochrome P4501A1, glutathione S-transferase Ya1, and UDP-glucuronosyltransferase 1*6 are apparently potentiated two- to fourfold upon inclusion of glucocorticoids in the media to activate the glucocorticoid receptor and further, that the receptor antagonist RU 38486 reverses these phenomenon. NAD(P)H:quinone oxidoreductase and aldehyde dehydrogenase 3 gene expression were repressed 70-80% by glucocorticoids in cultured hepatocytes through a glucocorticoid receptor-mediated process as well. The effect of glucocorticoid concentration on PAH induction of glutathione S-transferase Ya1 subunit for glucocorticoids was biphasic, but at physiological concentrations gene expression was repressed to approximately 20-40% of control. At supraphysiological concentrations, glucocorticoids alone induced expression two- to threefold and potentiated the PAH-inducible expression of the Ya1 subunit gene. Subsequent work in our laboratory has focused on defining the molecular basis of this hormonal regulation, specifically elucidating responsive elements responsible for the action of the glucocorticoid receptor and the mechanisms by which some of these genes are positively regulated and others are negatively regulated.

Effects of arsenite treatment on NAD(P)H:quinone acceptor oxidoreductase activity in liver, lung, kidney, and heart of the rat. Comparison to induction by the polyaromatic hydrocarbon, beta-naphthoflavone.

Arsenite is a potent toxin, a carcinogen, and an inducer of heat shock proteins. In this study we found that arsenite is also a novel inducer of NAD(P)H:quinone acceptor oxidoreductase (QOR) [EC 1.6.99.2] in both liver and kidney. The increases in activity were unlinked to those caused by prior treatment with the polyaromatic hydrocarbon inducer, beta-naphthoflavone suggesting different mechanisms of induction. A single dose of sodium arsenite (75 mumol/kg sc) caused a 4-fold and 2-fold increase in activity in kidney and liver, respectively, whereas beta-naphthoflavone (60 mg/kg ip once daily for 4 days) caused a 10-fold and 4.7-fold increase in kidney and liver, respectively. This is the first study of a metalloid inducing QOR activity. Arsenite is chemically unlike any other inducer described for QOR, which include phenolic antioxidants and Michael acceptors, polyaromatic hydrocarbons, and hydrogen peroxide. Arsenite also increased glutathione S-transferase [EC 2.5.1.18] activity in rat kidney. Arsenite could be inducing QOR in liver and kidney and the glutathione S-transferase activity in kidney by an oxidant stress mechanism.

Microsomal cytochrome P450 1A1 dependent monooxygenase activity in guinea pig heart: induction, inhibition, and increased activity by addition of exogenous NADPH-cytochrome P450 reductase.

Characterization of cytochrome P450 1A1 dependent monooxygenases in guinea pig heart revealed low rates of 7-ethoxyresorufin O-deethylation, which are markedly increased (20-fold) by treatment with beta-naphthoflavone, a polycyclic aromatic hydrocarbon. Both 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation were found to be approximately 4-fold higher in microsomes prepared from the ventricle than the atrium of beta-naphthoflavone-induced guinea pigs. The low rates of 7-ethoxyresorufin O-deethylation in cardiac microsomes were due, at least in part, to a deficiency of the flavoprotein NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase dramatically increased 7-ethoxyresorufin O-deethylation in cardiac microsomes of guinea pigs, before and after treatment with beta-naphthoflavone. N-Benzyl-1-aminobenzotriazole, a suicide substrate of cytochrome P450 1A1 in guinea pig, was able to inhibit almost all of the 7-ethoxyresorufin O-deethylase and 7-methoxyresorufin O-demethylase activities in polycyclic aromatic hydrocarbon induced guinea pig heart (88 and 71%, respectively), suggesting that cytochrome P450 1A1 coupled to NADPH--cytochrome P450 reductase in these microsomes inactivates itself by a suicidal mechanism. Addition of alpha-naphthoflavone, an inhibitor of cytochrome P450 1A isozymes, to cardiac microsomes from beta-naphthoflavone-induced guinea pigs resulted in greater than 95% inhibition of 7-ethoxyresorufin O-deethylase activity. The biological significance of these low levels of cytochrome P450 1A1 monooxygenase activity in guinea pig heart and their induction by polycyclic aromatic hydrocarbons are not currently understood.

Effects of acute sodium arsenite administration on the pulmonary chemical metabolizing enzymes, cytochrome P-450 monooxygenase, NAD(P)H:quinone acceptor oxidoreductase and glutathione S-transferase in guinea pig: comparison with effects in liver and kidney.

Tissue specific changes in the cytochrome P-450 (P-450) monooxygenase system were observed following a single subcutaneous dose of sodium arsenite (75 mumol/kg), a known inducer of stress proteins. P-450 monooxygenase activities were assayed with several isozyme selective substrates; 7-ethoxyresorufin, 7-pentoxyresorufin, 4-aminobiphenyl and erythromycin. Both tissue selective and isozyme selective changes in monooxygenase activity were noted. For example, the rate of 4-aminobiphenyl N-hydroxylation (ABH) was increased by arsenite administration in lung but not in liver. Arsenite inhibited 7-ethoxyresorufin O-deethylation (ERF) in all tissues of control animals, but to a lesser extent in lung. However, increases of ERF activity occurred after arsenite treatment in lung of beta-naphthoflavone (beta NF)-treated guinea pigs whereas arsenite decreased ERF activities in the kidney and liver of these animals. These complex effects on ERF activity may in part be modulated by induction of heme oxygenase, whose activity was increased 2.5-3.5-fold in these organs by arsenite. The highest heme oxygenase activity was found in kidney with lower activities being present in liver and lung, respectively. These data are consistent with the decreased P-450 content observed in kidney and liver microsomes of arsenite treated guinea pigs. On the other hand there was either no change or a slight increase (about 2-fold) in the pulmonary microsomal P-450 content of these animals. A complex pattern of induction for the non-heme, Ah locus associated enzyme, NAD(P)H:quinone acceptor oxidoreductase (QOR) was also observed. With menadione as substrate arsenite treatment increased QOR activity in all tissues studied. However, with dichlorophenolindophenol (DCPIP) as substrate a significant arsenite effect was observed only in the kidney. Significant differences between the QOR substrates were also observed in beta NF-treated guinea pigs and control animals. Our results are consistent with the presence of more than one form of QOR in the guinea pig. Arsenite treatment also caused an increase in glutathione S-transferase activity, with 2,4-dinitro-1-chlorobenzene (DNCB) as substrate, of guinea pig kidney but not liver or lung.