Oxathiolene oxides: a novel family of compounds that induce ferritin,glutathione S-transferase,and other proteins of the phase II response

Compounds that induce the synthesis of cytoprotective phase II enzymes have shown promise as cancer chemopreventive agents. Although chemically diverse, phase II enzyme inducers are capable of participating in Michael reaction chemistry. We have synthesized a novel class of organosulfur compounds, termed oxathiolene oxides (OTEOs). Based on their chemical properties, we hypothesized that these compounds could function as phase II enzyme inducers. Northern blot analysis showed that oxathiolene oxides induce the phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), and ferritin H and L mRNA in a concentration-dependent fashion in a normal embryonic mouse liver cell line, BNLCL.2. OTEO-562 (3-cyclohexenyl-4-methyl-1,2-oxathiol-3-ene-2-oxide) was the strongest inducer. Western blot analysis demonstrated that GST-alpha and ferritin H protein levels were also induced in cells treated with OTEO-562, as was total GST and NQO1 enzyme activity. Further, induction of NQO1 activity by OTEO-562 was equivalent in aromatic hydrocarbon (Ah) receptor wild-type and Ah receptor mutant cell lines, suggesting that oxathiolene oxides activate phase II enzymes by an Ah receptor-independent mechanism. Consistent with this observation, OTEO-562 failed to induce cytochrome P450 1A1 mRNA. These results suggest that oxathiolene oxides may merit further investigation as candidate chemopreventive agents.     

Modulation of aryl hydrocarbon receptor-regulated gene expression by arsenite, cadmium, and chromium

Aryl hydrocarbon receptor (AhR) ligands and heavy metals are environmental co-contaminants and their molecular interaction may disrupt the coordinated regulation of AhR-dependent phase I and II drug metabolizing enzymes. To determine the effect of heavy metals on the AhR-regulated genes: cytochrome P4501A1 (Cyp1a1), NAD(P)H: quinone oxidoreductase (QOR) and glutathione S-transferase Ya (GST Ya), murine hepatoma Hepa 1c1c7 cells were treated with increasing concentrations of As3+ (1-10 microM), Cd2+ (1-25 microM) and Cr6+ (1-25 microM) with or without the AhR ligands: 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1 nM), 3-methylcholanthrene (0.25 microM), beta-naphthoflavone (10 uM), or benzo[a]pyrene (1 microM). Our results show that AhR ligands alone and As3+ or Cd2+ alone increased the catalytic activities and mRNA levels of all AhR-regulated genes. When metals were co-administered with an AhR ligand, all three metals inhibited the induction of Cyp1a1 activity by the AhR ligands but potentiated its mRNA and protein expression. In addition, all metals enhanced QOR and GST Ya at the activity and mRNA levels but modulated their induction by AhR ligands in a concentration, metal, and AhR ligand-dependent manner. Generally, Cr6+ inhibited while As3+ and Cd2+ potentiated the induction of QOR and GST Ya activities and mRNA levels. The three metals enhanced the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. These results show that the ability of metals to alter the capacity of AhR ligands to induce the bioactivating phase I and the detoxifying phase II enzymes will influence the carcinogenicity and mutagenicity of the AhR ligands.     

Effects of aqueous extract of Ruta graveolens and its ingredients on cytochrome P450, uridine diphosphate (UDP)-glucuronosyltransferase,and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-quinone oxidoreductase in mice

Ruta graveolens (the common rue) has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes, effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP), uridine diphosphate (UDP)-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H):quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg) induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(P)H:quinone oxidoreductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (F_mix) increased Cyp2b activities in males. The mixture of rutin and F_mix increased Cyp1a and Cyp2b activities. These results revealed that rutin and F_mix contributed at least in part to the P450 induction by rue.     

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.     

Comparison of the induction of a 4S beta-naphthoflavone-binding protein, cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in beta-naphthoflavone-treated rats

A profound induction of a 4S beta-naphthoflavone (BNF)-binding protein, cytochrome P450 1A1 (CYP1A1) and NAD(P)H:quinone oxidoreductase (NQO1) activities was determined in the livers of Sprague-Dawley rats following intraperitoneal administration of BNF. Time-course of this induction differed for CYP1A1 and NQO1 activities, suggesting independent regulation of the phase I and II enzymes of xenobiotic metabolism. Time-course of the induction of CYP1A1 and BNF-binding activities was similar, suggesting that regulation of a 4S BNF- binding protein is associated with that of the CYP1A1 enzyme activity. The BNF specific binding to a 4S protein was inhibited by exogenous (BNF) and endogenous (indirubin and indigo) ligands for the aryl hydrocarbon receptor.     

Metabolic enzyme induction by HepG2 cells exposed to oxygenated and nonoxygenated polycyclic aromatic hydrocarbons

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) such as polycyclic aromatic quinones and polycyclic aromatic ketones as well as polycyclic aromatic hydrocarbons (PAHs) are abundant in the atmospheric environment. In this study, mRNA induction of six metabolic enzymes including P4501A1, 1A2, and 1B1, aldo-keto reductase 1C1 (AKR1C1), NAD(P)H-dependent quinone oxidoreductase 1 (NQO1), and glutathione S-transferase M1 (GSTM1) were examined in detail in human hepatoma (HepG2) cells exposed to environmentally relevant 13 PAHs and seven oxy-PAHs. Most PAHs such as benzo[a]pyrene (B[a]P) showed significant induction of P4501A1 and 1A2 mRNA, while induction by oxy-PAHs such as 5,12-naphthacenequinone (NCQ) and 11H-benzo[b]fluoren-11-one (B[b]FO) occurred less strongly. AKR1C1 mRNA was significantly induced by oxy-PAHs, 11H-benzo[a]fluoren-11-one (B[a]FO), NCQ, cyclopenta[cd]pyren-3(4H)-one (CPPO), and B[b]FO and also by P450s-inducing PAHs such as B[a]P, benzo[k]fluoranthene (B[k]FA), and dibenz[a,h]anthracene (DB[a,h]A). Both chemical-dependent and time-dependent induction patterns of NQO1 mRNA were of the mixed types of P4501A1 and AKR1C1. The tendency for the decrease of GSTM1 mRNA was observed when exposed to PAHs B[a]P and B[k]FA.     

Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: crosstalk through C/EBPs

Regulation of the rat glutathione S-transferase A2 (GSTA2) gene by glucocorticoids is biphasic in its concentration dependence to glucocorticoids, with concentrations of 10-100 nM repressing gene activity (GR-dependent), and concentrations above 1 microM increasing transactivation (PXR-dependent) in adult rat hepatocytes or transient transfection assays. Over-expression of either C/EBP alpha or beta negatively regulates basal and inducible expression of a 1.65 Kb GSTA2 luciferase reporter, and synergizes the response to glucocorticoids (GC). C/EBP responsive elements have been identified in the GSTA2 5'-flanking sequence, associated with the palindrominic Glucocorticoid Responsive Element (GRE), the Ah receptor response elements, and the antioxidant response element. In reporters lacking the palindromic GRE, negative regulation by GC is observed only when C/EBP alpha is co-expressed. Co-transfection of C/EBP alpha/beta induced gene expression of the GSTA2 XRE reporter, but negatively regulated the GSTA2 ARE-reporter. In contrast, the ARE from the rat NAD(P)H quinone oxidoreductase gene was induced by co-transfection of C/EBPs, but was still negatively regulated by GC. PXR-induction of the GSTA2 reporter was partially ablated by co-transfection of C/EBP alpha and enhanced by co-transfection of C/EBPbeta. We conclude that C/EBP alpha and beta are involved in GC-dependent repression of GSTA2 gene expression and ARE sequences that bind C/EBPs appears to be critical for these responses.     

The effects of tannic acid on cytochrome P450 and phase II enzymes in mouse liver and kidney

Tannic acid, a naturally occurring plant polyphenol, was shown to decrease the mutagenicity and/or carcinogenicity of several amines derivatives and polycyclic aromatic hydrocarbons in rodents. The aim of this study was to evaluate the effect of tannic acid on the activities of murine cytochrome P450 and phase II enzymes. The activities of ethoxyresorufin-O-deethylase (EROD), methoxyresorufin-O-demethylase (MROD), p-nitrophenol hydroxylase (PNPH), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UDPGT) and NAD(P)H:quinone oxidoreductase (NQO1) were measured in the liver and kidney microsomes of female Swiss mice treated intraperitoneally (i.p.) with tannic acid in the dose range of 20-80 mg/kg. At the highest dose, tannic acid decreased the activities of EROD and MROD by 25-28% in mouse liver, while the activity of both hepatic and renal PNPH was reduced by approximately 50% as result of treatment. Moreover, Western blot analysis with CYP2E1 specific antibody showed a significant decrease in the levels of hepatic CYP2E1 in tannic acid treated animals. This polyphenol affected also the phase II enzymes in both tissues examined. The activity of GST was elevated in kidneys, but reduced in livers of the animals treated with tannic acid. The most striking effect was the inhibition of hepatic NQO1. The effect was dose dependent and almost 90% inhibition was observed after the treatment with tannic acid at the dose of 60 and 80 mg/kg. The same treatment caused the approximately 60% inhibition of renal NQO1. These results indicate that tannic acid, beside of scavenging active metabolites of chemical carcinogens, can change their metabolism by modulating the enzymes involved in xenobiotics activation and/or detoxification pathways.     

Changes in the expression of cytochrome P450 2E1 and the activity of carcinogen-metabolizing enzymes in Schistosoma haematobium-infected human bladder tissues

The bioactivation of N-nitrosamines and polycyclic aromatic hydrocarbons (PAHs) is mediated primarily by the mixed-function oxidase system, which includes dimethylnitrosamine N-demethylase I, arylhydrocarbon [benzo(a)pyerne] hydroxylase, cytochrome P450, cytochrome b(5), and ethoxycoumarin deethylase. Most of carcinogens and xenobiotics are conjugated and detoxified by phase II drug-metabolizing enzymes such as glutathione S-transferase. The present study showed the influence of Schistosoma haematobium on the activity of the above-mentioned enzymes in 13 schistosome-infected human bladder tissues compared with those of 15 schistosome-free samples. The contents of cytochrome P450 and cytochrome b(5) were increased in the bladder tissues by 48 and 69%, respectively. Moreover, the activities of dimethylnitrosamine N-demethylase I and arylhydrocarbon hydroxylase, ethoxycoumarin O-deethylase, ethoxyresourfin O-deethylase, and pentoxyresorufin O-pentoxyresorufin were increased by 75, 159, 49, 63 and 44%, respectively. The signal intensity for cytochrome P450 2E1 was greatly increased over the control. Also, the activity of glutathione S-transferase was increased by 89%. On the other hand, the activity of glutathione reductase and the level of reduced glutathione were decreased by 40 and 57%, respectively. Interestingly, the level of free radical, thiobarbituric acid reactive substance, was increased in the schistosome-infected human bladder tissues by 125%. The present study clearly demonstrated that S. haematobium changes the activity of carcinogen-metabolizing enzymes. We conclude that S. haematobium could enhance the carcinogenicity of polycyclic aromatic hydrocarbons (e.g. benzo(a)pyrene) and N-nitrosamines (e.g. dimethylnitrosamine) through induction of their corresponding bioactivating enzymes in human bladder tissues.     

Role of cytochrome P450 and glutathione S-transferase alpha in the metabolism and cytotoxicity of trichloroethylene in rat kidney

The toxicity and metabolism of trichloroethylene (TRI) were studied in renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. TRI was slightly toxic to both PT and DT cells, and inhibition of cytochrome P450 (P450; substrate, reduced-flavoprotein:oxygen oxidoreductase [RH-hydroxylating or -epoxidizing]; EC 1.14.14.1) increased TRI toxicity only in DT cells. In untreated cells, glutathione (GSH) conjugation of TRI to form S-(1,2-dichlorovinyl)glutathione (DCVG) was detected only in PT cells. Inhibition of P450 transiently increased DCVG formation in PT cells and resulted in detection of DCVG formation in DT cells. Formation of DCVG in PT cells was described by a two-component model (apparent Vmax values of 0.65 and 0.47 nmol/min per mg protein and Km values of 2.91 and 0.46 mM). Cytosol isolated from rat renal cortical, PT, and DT cells expressed high levels of GSH S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) alpha (GSTalpha) but not GSTpi. Low levels of GSTmu were detected in cortical and DT cells. Purified rat GSTalpha2-2 exhibited markedly higher affinity for TRI than did GSTalpha1-1 or GSTalpha1-2, but each isoform exhibited similar VmaX values. Triethyltinbromide (TETB) (9 microM) inhibited DCVG formation by purified GSTalpha-1 and GSTalpha2-2, but not GSTalpha1-2. Bromosulfophthalein (BSP) (4 microM) only inhibited DCVG formation by GSTalpha2-2. TETB and BSP inhibited approximately 90% of DCVG formation in PT cytosol but had no effect in DT cytosol. This suggests that GSTalpha1-1 is the primary isoform in rat renal PT cells responsible for GSH conjugation of TRI. These data, for the first time, describe the metabolism of TRI by individual GST isoforms and suggest that DCVG feedback inhibits TRI metabolism by GSTs.     

Inducible bilirubin-degrading system of rat liver microsomes: role of cytochrome P450IA1

The ability of 3,3',4,4'-tetrachlorobiphenyl to stimulate bilirubin degradation by liver microsomes from rats treated with a polycyclic aromatic hydrocarbon-type inducer has been confirmed and extended to another planar biphenyl, 3,3',4,4',5,5'-hexachlorobiphenyl. The following evidence indicates the involvement of an inducible cytochrome P450 isoenzyme in this reaction, with a role, specifically, for cytochrome P450IA1. (a) The biphenyl-dependent bilirubin degradation and 7-ethoxyresorufin O-deethylase (EROD) activity were both markedly inhibited by a monoclonal antibody raised against cytochrome P450IA1; the two dose-inhibition curves were essentially superimposable, with maximum inhibition observed for both activities at a ratio of antibody to total cytochrome P450 of about 1. (b) Treatment of rats with 3-methylcholanthrene increased both EROD activity and biphenyl-dependent bilirubin degradation not only in the liver (where both cytochromes P450IA1 and P450IA2 are inducible) but also in the kidney (where only induction of cytochrome P450IA1 has been reported), with similar ratios of the two enzymatic activities in both tissues. (c) With carbon tetrachloride and 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,4-dimethyl pyridine as selective suicide substrates of members of the cytochrome P450IA subfamily, the biphenyl-dependent degradation of bilirubin showed a good correlation with cytochrome P450IA1, determined both as EROD activity and as an immunoreactive band on immunoblotting. These findings implicate cytochrome P450IA1 in the alternative pathway of bilirubin disposal, which can be stimulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin in Gunn rats, and also help substantiate the hypothesis that interaction of a polyhalogenated aromatic compound with the induced cytochrome may initiate an oxidative mechanism leading to oxidation of target molecules in the cell, one of which is bilirubin.     

A possible modulatory influence of melatonin on representative phase I and II drug metabolizing enzymes in 9,10-dimethyl-1,2-benzanthracene induced rat mammary tumorigenesis.

The oncosuppressive effect of melatonin on 9,10-dimethyl-1,2-benzanthracene (DMBA) induced rat mammary tumorigenesis led us to assess its possible modulatory influence on representative hepatic and mammary drug metabolizing enzymes in DMBA treated female Holtzman rats, reared in short and long photoperiods. Melatonin treated rats in either photoperiod showed a significant induction in hepatic and mammary levels of glutathione (GSH) and cytosolic activities of glutathione S-transferase (GST) when compared with the corresponding controls, along with a significant drop in hepatic microsomal contents of cytochromes b5 and P450. This induction of GSH and GST, and depletion of cytochromes b5 and P450 by melatonin may possibly be related to its anticarcinogenic potential in this tumor model.     

Persistent induction of hepatic and pulmonary phase II enzymes by 3-methylcholanthrene in rats.

We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P450 (CYP)1A expression for up to 45 days by mechanisms other than persistence of the parent MC (Moorthy, J. 2000. Pharmacology. Exp. Ther. 294, 313-322). The CYP1A genes are members of the Ah gene battery that also encode CYP1B1 and phase II enzymes such as glutathione S-transferase (GST-alpha), UDP glucuronyl transferase (UGT)1A, NAD(P)H (nicotinamide adenine dinucleotide phosphate, reduced):quinone oxidoreductase I (NQO1), aldehyde dehydrogenase (ALDH), etc. Therefore, in this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1B1 and phase II genes, which are in part regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 mumol/kg), ip, once daily for 4 days, and expression of CYP1B1 and several phase II (e.g., GST-alpha, NQO1) genes and their corresponding proteins were determined in lung and liver. The major finding was that MC persistently induced (3- to 10-fold) the expression of several phase II enzymes, including GST-alpha, NQO1, UGT1A1, ALDH, and epoxide hydrolase in both tissues for up to 28 days. However, MC did not elicit sustained induction of CYP1B1. Our results thus support the hypothesis that MC elicits coordinated and sustained induction of phase II genes presumably via persistent activation of the AHR, a phenomenon that may have implications for chemical-induced carcinogenesis and chemopreventive strategies in humans.     

Intrinsic acute toxicity and hepatic enzyme inducing properties of the chemoprotectants indole-3-carbinol and 5,10-dihydroindeno[1,2-b]indole in mice

Indole-3-carbinol (I-3-C) and 5,10-dihydroindeno[1,2-b]indole (DHII) have been shown to be protective against carbon tetrachloride and other chemicals that cause hepatic toxicity. In part, this protection appears to be afforded by the ability of these compounds to act as antioxidants, with DHII having much the greater efficacy. In order to understand the mechanisms of chemoprotection, as well as the potential for therapeutic and pharmaceutical use in humans, the antioxidants I-3-C and DHII were examined for their intrinsic acute toxicity, and their hepatic enzyme inducing properties in mice. The results were compared with those of the well characterized agent phenobarbital. Following treatment by gavage for 10 days with 50 mg compound/kg body weight, I-3-C produced modest (10-50%) increases in hepatic cytochrome P-450, aminopyrine N-demethylase, UDP-glucuronosyl transferase (UDPGT) and glutathione S-transferase (GST), and a four-fold increase in NAD(P)H: (quinone acceptor) oxidoreductase (quinone reductase) activity. DHII did not alter oxidative enzyme activities, but increased GST and UDPGT by about 50%, and quinone reductase over five-fold. In the acute toxicity studies, DHII produced no observable 24-hr acute toxicity up to 4 g/kg body weight, except for a slight decrease in haematocrit. However, I-3-C exhibited a dose-dependent toxicity above 100 mg/kg body weight, including a decrease in hepatic reduced glutathione after 2 hr and severe neurological toxicity, and the release of liver enzymes to the plasma at 24 hr. We conclude, on the basis of the superior antioxidation efficacy of DHII, its enzyme-inducing properties, and intrinsic toxicity, that DHII or cogeners thereof have great potential as chemoprotective or therapeutic agents. However, I-3-C does not have such potential.     

Sex-specific biotransformation and detoxification after xenobiotic exposure of primary cultured hepatocytes of European flounder (Platichthys flesus L.)

Sex-specific effects of sublethal concentrations of known effective pro-oxidants such as 100,200 and 400 microM benzo[a]pyrene (B[a]p), 50 microM nitrofurantoin (NF) and 100 microM hydrogen peroxide (H2O2) on biotransformation pathways were studied in isolated hepatocytes of immature female and male European flounder (Platichthys flesus L.). Cell responses were assessed at the level of: (1) stress induction as measured by formation of reactive oxygen species (ROS), mainly superoxide radicals, and induction of cytochrome P450 (CYP450) biotransformation activity; (2) cellular antioxidant defences, both non-enzymatic (reduced glutathione) and enzymatic (DT-diaphorase (DTD) or quinone oxidoreductase, EC 1.6.99.2); (3) detoxification (aldehyde dehydrogenase (ALDH), EC 1.2.1.3); and (4) cellular damage as measured by reduced lysosomal membrane stability and cell death. As there is increasing evidence that 17-beta-estradiol interferes with certain pathways of xenobiotic biotransformation, we additionally tested the effects of different concentrations of 17-beta-estradiol (0.2-10 microM) alone and 17-beta-estradiol (1 microM) in combination with 100 microM B[a]p. Parameters were monitored after 1 and 9 days of exposure by quantitative image analysis of chromogenic or fluorogenic reaction products. Our study revealed sex-dependent differences in cellular stress responses. In hepatocytes of female flounder, biotransformation was slower and the capacity of non-enzymatic antioxidant defences and detoxification of toxic aldehydes was lower than in males. Additional administration of 17-beta-estradiol enlarged these differences between the sexes with respect to biotransformation activity and antioxidant defence in xenobiotic-induced injury. These findings may explain the higher susceptibility of female flounder to toxic and carcinogenic compounds in the marine environment.     

High affinity phenacetin O-deethylase is catalysed specifically by cytochrome P450d (P450IA2) in the liver of the rat

Phenacetin is metabolized primarily by O-deethylation to paracetamol (POD activity), a reaction catalysed by cytochrome P450. The high affinity component of POD activity is inducible in rat liver by treatment of the animals with polycyclic aromatic hydrocarbons. Following treatment with hydrocarbons such as 3-methylcholanthrene (MC) and isosafrole (ISF) both cytochromes P450c (P450IA1) and P450d (P450IA2) are also induced in rat liver. Studies with the reconstituted enzymes have shown that both forms of P450 catalyse phenacetin O-deethylation at rates that exceeded that of the high affinity component of activity of hepatic microsomal preparations from 3-methylcholanthrene-treated rats (at 4 microM phenacetin: P450c, 440 +/- 40 pmol/nmol/min; P450d, 1030 +/- 10 pmol/nmol/min; microsomal fraction, 163 pmol/mg/min). Specific inhibitory antibodies (both monoclonal and monospecific polyclonal) were used to define the specificity of microsomal POD activity. These studies have shown that hepatic high affinity POD activity is exclusively catalysed by cytochrome P450d in both untreated rats and in rats pretreated with MC.