Mixed Function Oxidase Activity Research Articles

Characterization of microsomal oxidative activities in a wild-type and in a DDT resistant strain of Drosophila melanogaster

Resistance of a laboratory selected DDT strain of Drosophila melanogaster (RalDDT R) has been found to be monofactorial and correlated to an increased level of activity of the cytochrome P450-dependent mixed function oxidase (MFO). Both strains metabolize DDT and deltamethrin via MFO activity. However, the resistant strain does it more rapidly. The amount of DDT metabolites, including kelthane, bis-4-chlorophenyl acid, bis-4-chlorophenyl-ethanol, and 1,1-bis ( p-chlorophenyl)2,2-dichloroethane, is approximately 9-fold greater with RalDDT R microsomes than with the wild-type strain Raleigh (Ral). Production of deltamethrin metabolites is 2.7-fold higher within the resistant strain. As compared to insecticides, lauric acid and the two steroids used as substrates in this study present many more sites for MFO metabolic action. Lauric acid is hydroxylated on positions 11 and 12 by both strains, but the amount of metabolites formed is 10-fold higher with RalDDT R microsomes. The 2,22-dideoxyecdysone is converted to two polar metabolites when incubated with RalDDT R microsomal preparations. These unidentified metabolites are neither 2-deoxyecdysone nor ecdysone. Also reported for the first time is the metabolization of testosterone by insect microsomes, which gives 13 oxiderivatives formed at different rates, depending on the strains.

Aminoglutethimide enzyme induction: pharmacological and endocrinological implications

Aminoglutethimide is an aromatase inhibitor that is successfully used for endocrine treatment of advanced breast cancer. This drug also stimulates the activity of hepatic mixed-function oxidases, increasing the metabolism of several drugs, including warfarin, digitoxin, antipyrine and theophylline. It also increases the plasma clearance rate of oestrone sulphate. As this oestrogen may be an important substrate for tumour cells, stimulation of oestrone sulphate metabolism may be a component of the mechanism of action of aminoglutethimide.

Multiple effects of 3,4,5,3′,4′,5′-hexachlorobiphenyl administration on hepatic cytochrome P450 isozymes and associated mixed-function oxidase activities in rainbow trout

Multiple effects of 3,4,5,3′,4′,5′-hexachlorobiphenyl administration on hepatic cytochrome P450 isozymes and associated mixed-function oxidase activities in rainbow trout

Effect of genetic obesity and experimental diabetes on hepatic microsomal mixed function oxidase activities

In male rats, genetic obesity and experimental diabetes are associated with altered activities of several of the hepatic microsomal P-450 isozymes concerned with steroid and xenobiotic oxidation. The present study examined the roles of insulin and ketonaemia in effecting these changes. In obese male Zucker rats, androstenedione 6 beta-, 16 alpha- and 16 beta-hydroxylase activities (mediated by P450PCN-E, P-450UT-A and P450PB-B, respectively) were significantly decreased to 21%, 20% and 43% of lean control. Obesity was also associated with a significant decrease in the activities of N-nitrosodimethylamine demethylase (P-450j) and aniline p-hydroxylase to about 70%. A similar decrease in total microsomal P-450 was also observed. Androstenedione 7 alpha-hydroxylase activity (mediated by P-450UT-F) was unchanged in these animals. In streptozotocin-induced diabetic male Wistar rats, androstenedione 7 alpha- and 16 beta-hydroxylase activities were significantly elevated to 230% and 270% of control, respectively. Significant increases in the rates of N-nitrosodimethylamine demethylase and aniline p-hydroxylase were also noted in diabetic rat liver. In contrast, the activity of P-450UT-A was reduced to 30% of control and P-450PCN-E-specific 6 beta-hydroxylation was unchanged. Control of the diabetic state with insulin treatment reversed all the changes in P-450-mediated activities. Significant correlations were found between serum concentrations of insulin and catalytic activities of P-450PB-B (rho = -0.46), P-450UT-F (rho = -0.65) and P-450j (rho = -0.71). Positive correlations of the same magnitude were also found between these mixed function oxidase activities and beta-hydroxybutyrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic microsomal cytochrome P450 system during experimental hookworm infection

Experimental infection of golden hamsters with the hookworm, Ancylostoma ceylanicum, caused a profound decline in the hepatic microsomal cytochrome P450 content. Concomitant decrease was also noticed in aminopyrine N-demethylase and benzo[ a]pyrene hydroxylase activities. However, aniline hydroxylase activity was only marginally elevated during the infection. Microsomal markers, viz., cytochrome b 5, NADH-cytochrome- c reductase, and glucose-6-phosphatase, were not significantly altered. Hepatic tissue exhibited an accumulation of lipids, especially phospholipids, triglycerides, and cholesterol, resulting in fatty necrosis around the central vein region. Isolated hepatic microsomes showed a decrease in phosphatidylcholine content. Impairment in hepatic mixed function oxidase (MFO) activities was further confirmed by prolongation in hexobarbital sleeping time and zoxazolamine-induced paralysis. The hepatic MFO system of A. ceylanicum-infected hamsters responded qualitatively and quantitatively in a manner similar to that of control hamsters, upon stimulation with selective chemical inducers like phenobarbitone and 3-methylcholanthrene. Kinetic and in vitro substrate binding studies revealed that for aminopyrine the substrate affinity and the maximum enzyme activity ( V max) were decreased, while for aniline the binding affinity was decreased and the binding capacity was enhanced. Results indicate specific/selective impairment of the hepatic microsomal cytochrome P450 system during hookworm infection and may have many practical implications in toxicology and pharmacology.

The Influence of Detoxifying Enzymes on Insecticide Tolerance in Honey Bee Colonies (Hymenoptera: Apidae)

Colonies of the honey bee, Apis mellifera L., showed variation in tolerance to the insecticides diazinon, propoxur, aldrin, and carbaryl. Tolerance to diazinon and propoxur was positively related to midgut mixed-function oxidase and glutathione transferase enzyme activities, but tolerance to aldrin was inversely related to these enzymes. Differences in tolerance to carbaryl were evident among colonies but were not related to enzyme activity levels. Linear regression models derived from these data may be used to predict colony tolerance from enzyme activity and may enable the selection of tolerant strains of bees that could maintain efficient pollination in areas of extensive insecticide use.

Alterations in xenobiotic metabolizing enzymes in brain and liver of rats coexposed to endosulfan and malathion

The effects of endosulfan (3 mg kg-1 body wt., i.p.) and malathion (30 mg kg-1 body wt.) and their coexposure on rat hepatic and brain xenobiotic metabolizing enzymes were investigated. Endosulfan was found to induce aminopyrine-n-demethylase (81%) and aniline hydroxylase (59%) activities significantly in liver and to a lesser extent in brain. Malathion treatment induced malathion carboxylesterase activity in both liver (50%) and brain (22%), significantly depleted liver glutathione (35%) content with stimulation of glutathione-S-transferase (50%) and inhibited the activity of mixed-function oxidases. In the coexposed animals, malathion's inhibitory influence on mixed-function oxidases and endosulfan's inhibitory effect on malathion carboxylesterase were found to dominate, while endosulfan potentiated the activity of glutathione-S-transferase significantly in liver (69%) and brain. A similar trend of alteration in coexposed brain was found, but to a lesser extent. A significant inhibition in brain acetylcholine esterase activity (42%) in the coexposed animals suggests that endosulfan may potentiate the toxicity of malathion by interfering with glutathione and carboxylesterase routes of malathion detoxification.

Influence of Hyperalimentation on Rat Hepatic Microsomal Fluidity and Function

Total parenteral nutrition with an amino acid-glucose solution has previously been shown to decrease rat hepatic drug metabolism compared with drug metabolic activity observed in rats receiving the same solution enterally and chow-fed animals. Because changes in membrane fluidity and lipid composition are reported to influence activity of a number of liver enzymes, effects of parenteral and enteral nutrition on hepatic microsomal membrane fluidity and lipid composition were assessed and compared with hepatic mixed-function oxidase activity. Both parenteral and enteral hyperalimentation produced a significant decrease in microsomal membrane fluidity (fluorescence anisotropy = 0.155 +/- 0.003 in both experimental groups versus 0.129 +/- 0.003 for microsomes from chow-fed animals). However, meperidine demethylase activity was significantly decreased compared with chow-fed experiments only in hepatic microsomes from parenterally hyperalimented animals, whereas ethoxyresorufin deethylase activity was significantly reduced only in the enteral-nutrition group. Inclusion of lipid in the parenterally administered hyperalimentation solution normalized microsomal membrane fluidity and lipid profile to those of chow-fed animals but did not increase hepatic meperidine demethylation. Both parenteral and enteral nutrition produce significant changes in physical state and lipid composition of rat hepatic microsomal membranes, but these changes are not responsible for the altered hepatic drug metabolism observed during hyperalimentation.

Correlation of azinphosmethyl resistance with detoxication enzyme activity in the light brown apple moth Epiphyas postvittana (Lepidoptera: Tortricidae)

Bioassay using diet incorporation of azinphosmethyl showed six strains of light brown apple moth to have varying levels of resistance to azinphosmethyl (3- to 15-fold) compared with three susceptible strains. Two of these strains were products of a selection and backcrossing program providing resistant insects that had a common genome with that of one of the susceptible strains. Individual male moths from each strain were tested for (i) glutathione S-transferase and (ii) non-specific esterase activity. Larvae were assayed for (iii) mixed function oxidase activity. Each enzyme group was assayed with two substrates. Significant positive regressions between LC 50 and enzyme activity were observed for all three groups: (i) r 2 = 0.84 for 3,4-dichloronitrobenzene conjugation, (ii) 0.64 for α-naphthyl acetate hydrolysis, 0.54 for α-naphthyl butyrate hydrolysis, and (iii) 0.88 for p-nitroanisole oxidation and 0.86 for aniline oxidation. No correlation with the LC 50 was found for 1-chloro-2,4-dinitrobenzene conjugation ( r 2 = 0.001). Activities of all three groups were significantly higher in the two backcross resistant strains compared to the susceptible parent strain ( P < 0.05). The gene(s) for high enzyme activity had thus been successfully transferred with the factor for resistance. The results suggest a close relationship between detoxication enzyme activity and azinphosmethyl resistance.

Localization of pulmonary carbonyl reductase in guinea pig and mouse: enzyme histochemical and immunohistochemical studies.

We studied the localization of carbonyl reductase (E.C. 1.1.1.184) in guinea pig and mouse lung by enzyme histochemistry and immunohistochemistry, using antibodies against the guinea pig lung enzyme which crossreacted with the lung enzymes of both animals. Carbonyl reductase activity was detectable in the bronchiolar epithelial cells of small airways and in alveolar cells. In the immunohistochemical staining for carbonyl reductase, the reaction was strongest in the non-ciliated bronchiolar cells (Clara cells) and was weak in the ciliated cells and type II alveolar pneumocytes. Injection of a single dose of naphthalene led to significant impairment of carbonyl reductase activity and of microsomal mixed-function oxidase activities in mouse lung, with a marked decrease in both activity and immunoreactive staining in the bronchiolar epithelial cells. The results indicate that carbonyl reductase is localized primarily in the Clara cells, which are known to be sites of pulmonary drug metabolism.

Inhibition of hepatocarcinogenesis in mice by dietary methyl donors methionine and choline

A dietary deficiency of methyl donors, the lipotropes methionine and choline, enhances the activity of hepatocarcinogens in rodents. To determine if the reverse is true, an excess of dietary choline, methionine, or both was fed to male mice given a carcinogenic dose of aflatoxin B1 (AFB1). Fifty weeks following the last dose of AFB1, all survivors were killed then examined for tumor incidence, and samples of nontumorous liver tissue were assayed for activities of mixed function oxidases (MFO). Survival was best in the high-methionine/high-choline group, with 36/38 surviving to termination of the study. Survival in the other groups was 35/38, 30/70, 33/38, and 34/37 in control with no AFB1, control with AFB1, groups with high methionine, and high choline, respectively. Combined adenoma/carcinoma incidence was 8/38, 30/37, 21/38, 20/37, and 10/38 in groups control with no AFB1, control with AFB1, high methione with AFB1, high choline with AFB1, and high choline and high methionine with AFB1, respectively. Cytochrome P450, cytochrome B5, cytochrome C, and ethylmorphine N-demethylase activities were all increased over controls, with most marked increases in the cytochrome P450 and ethylmorphine N-demethylase activities. The data presented here document a protective effect of dietary methyl donors on AFB1-induced hepatocarcinogenesis in mice probably acting, in part, via activation/detoxification mechanisms favoring an increased balance in detoxification of AFB1.

Characterization of 7-ethoxycoumarin- O-deethylase from malathion resistant and susceptible strains of Drosophila melanogaster

Mixed-function oxidase activity in a D. melanogaster strain carrying at least two closely linked malathion resistance genes on chromosome 3 was compared with the susceptible Canton S strain. The kinetics of O-deethylation of 7-ethoxycoumarin (7-ECD activity) with respect to pH, temperature, substrate and cofactor (NADPH) affinities and the response to metal salts of both strains were similar. The resistant strain had approx. 5-fold greater 7-ECD specific activity, and a parallel increase in total cytochrome P-450 content. Developmental stage, sex and nutritional state affected Drosophila 7-ECD activity. The intestine, fat body and Malpighian tubules contained the largest 7-ECD specific activity. Both susceptible and resistant strains had similar patterns of 7-ECD expression and differed only in total activity. In addition to more cytochrome P-450, the resistant strain had increased amounts of two microsomal, heme-staining polypeptides ( M r = 50 and 54 K after SDS-PAGE). The results suggest that the genetic change in the resistant strain involves the regulation of the Drosophila cytochrome P-450 system.

Biliary Excretion Appears Rate Limiting for Hepatic Elimination of Benzo[a]pyrene by Temperature-Acclimated Rainbow Trout

Biliary Excretion Appears Rate Limiting for Hepatic Elimination Of Benzo[a]pyrene by Temperature-Acclimated Rainbow Trout. CURTIS, L. R., FREDERICKSON, L. K., AND CARPENTER, H. M. (1990) Fundam. Appl. Toxicol. 15, 420–428. Previous work demonstrated that mixed function oxidase activities of hepatic microsomes from cold- and warm-acclimated rainbow trout were similar when assayed at temperatures to which fish were acclimated. This “ideal temperature compensation” was partially explained by constitutive differences in microsomes. In the work reported here, rainbow trout were acclimated at 10 or 18°C for 4 weeks and then ip injected with 10 μmol [3H] or [14C]benzo[a]pyrene (BP)/kg in one of two temperature regimens. First, fish were acclimated and exposed at the same temperature and killed after 4, 24, or 48 hr. Concentrations of [3H]BP equivalents in liver, bile, and fat but not in plasma, muscle, intestine, gill, or kidney increased with time. There were no differences in hexane or ethyl acetate extract-able [3H] or [14C]BP tissue concentrations in 10 and 18°C-acclimated fish exposed at their acclimation temperatures. At 24 hr after injection, biliary excretion of [3H]BP equivalents was about twofold higher at 18°C than at 10°C. Therefore, warmer temperature stimulated biliary excretion without a marked effect on in vivo BP metabolism. In the second regimen, 10 and 18°C-accli-matedfish were shifted to 14°C, injected with [3H] or [14C]BP 1 hr later, and killed after an additional 24 hr. There were no differences in tissue concentrations of total [3H]BP equivalents between acclimation groups at 14°C. However, the biliary concentration of [14C]BP not extracted by ethyl acetate was significantly higher in bile from 10°C-acclimated fish than from 18°C-acclimated fish when both groups were exposed at 14°C. In this case, in vivo BP metabolism was altered without coincident effects on biliary excretion. Aryl hydrocarbon hydroxylase assayed at 14°C was about threefold higher in hepatic microsomes from 10°C-acclimated fish than from 18°C-acclimated fish. Exposure temperature selectively modulated BP metabolism and biliary excretion in temperature-acclimated rainbow trout.

Differential localization of microsomal mixed function oxidase activity in periportal and perivenous hepatocytes isolated from rat liver

1. 1. The activity per mg of microsomal protein of aminopyrine N- demethylase was higher in perivenous (PV) than in periportal (PP) hepatocytes of rat, but when it was expressed per cytochrome P-450 content the difference in the activity was not significant. 2. 2. The activity of 7-ethoxycoumarin O-deethylase, when expressed per mg protein and per P-450 content, was significantly higher in PV than in PP cells. 3. 3. The activities of dimethylnitrosamine(DMNA) N-demethylase and aniline p-hydroxylase were not significantly different between two subpopulations of isolated hepatocytes when either expressed per mg protein or per P-450 content.

Determination and occurrence of AHH-active polychlorinated biphenyls, 2,3,7,8-tetrachloro-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran in Lake Michigan sediment and biota. The question of their relative toxicological significance

An analytical procedure has been developed for the determination of the 18 PCB congeners which are inducers of methylcholanthrene-like mixed function oxidase activity in animals and include the most toxic PCBs. Determinations of the toxic PCB congeners in samples of eggs of predatory fish and piscivorous birds of the Great Lakes and in Aroclor mixtures demonstrate that the apparent toxic potency of PCB residues in these samples is dominated by two congeners, 3,3′,4,4′,5- and 2,3,3′,4,4′-pentachlorobiphenyl. Furthermore, the analyses demonstrate that the potential toxicity of PCB residues can increase 5 to 10 fold as they reach upper levels of aquatic food chains and most often exceed the potential toxicity of chlorinated dibenzodioxins and furans in higher animals even in environments highly contaminated by the latter compounds.

Primary cultures of rabbit renal proximal tubule cells: II. Selected phase I and phase II metabolic capacities

Specific characteristics of cells vary as a function of time in culture. We have determined the stability of selected Phase I and Phase II biotransformation capacities in rabbit renal proximal tubule cells in primary culture. When grown in hormonally-defined medium, proximal tubule cells lost Phase I metabolic capacity. Cytochrome P-450 content and associated mixed-function oxidase activities present in kidney cortex microsomes were not detectable after 14 days in culture. Phase II glutathione-dependent metabolic functions were well retained in cultured cells compared with freshly isolated proximal tubules (FIPT). Cellular total glutathione content was 2.8 μg/mg protein in FIPT compared with approximately 10 μg/mg protein in stable confluent cultures. A higher total glutathione content of 20.6 μg/mg was noted in preconfluent cultures. The glutathione redox state was initially perturbed in FIPT with 37% of the total glutathione present found in its oxidized form. Tubule cells recovered to a normal ratio (6–13% of total glutathione in the oxidized form) while in culture. The glutathione S-transferase activity in 4-day-old cells in culture was reduced to 50% of the 4 U/mg protein level found in FIPT. No appreciable further decline in glutathione S-transferase activity was detected during 15 days in culture. The level of γ-glutamyl-transpeptidase (a brush-border enzyme necessary for glutathione uptake into proximal tubule cells) declined from 1499 mU/mg protein in homogenates of FIPT to 636 mU/mg in homogenates of 8-day-old cultured cells. A further decline in activity occurred during the next 7 days in culture. In conclusion, although Phase I metabolic functions were diminished in primary cultured rabbit proximal tubule cells, Phase II metabolic functions were retained at levels comparable with FIPT and well above those found in several established kidney cell lines.

The use of single sample clearance estimates to probe hepatic drug metabolism: handprinting the influence of cigarette smoking on human hepatic drug metabolism

1. Conditions were examined under which estimates of drug clearance made from a single measurement of plasma concentration effectively represented multiple-sample estimates of clearance for quinidine, valproic acid, unbound valproic acid, and lorazepam. When plasma concentrations were measured at various post-dose times, both individual and mean values of single-sample clearance estimates, CL, corresponded closely to multiple-sample clearance estimates. Best post-dose sampling times were: quinidine, 8 h; valproic acid, 24 h; and lorazepam, 24 h. 2. Single-sample clearance estimates, CL, were calculated for seven drugs employed as probes of human hepatic drug-metabolizing enzymes. Valproic acid was used to probe microsomal and peroxisomal beta-oxidase activity; antipyrine, phenytoin, quinidine, carbamazepine, and theophylline were used as probes of hepatic mixed-function oxidases (MFO), and lorazepam as a probe for UDP-glucuronosyltransferase activity. 3. A clearance index (CI, namely probe CL for smokers divided by probe CL for non-smokers) was calculated for each probe. The effect of cigarette smoking (and presumably polycyclic aromatic hydrocarbon exposure) on all probe CL values was consolidated and plotted as the logarithm of the CI to produce a handprint of drug metabolizing enzyme activity for cigarette smokers. 4. Only theophylline CL was significantly faster among smokers than non-smokers (P less than 0.01). 5. We conclude that the use of multiple probes of MFO activity when given in a single-dose, single-sample protocol for structuring handprints represents a minimally invasive and useful approach to characterize xenobiotic-mediated effects on hepatic MFO.

Freshwater fish cytochrome P450-dependent enzymatic activities: A chemical pollution indicator

Fish mixed function oxidase activities were measured during four sampling campaigns in the river Durance in the southeast of France. Six hundred fish belonging to four species were caught for this study; the use of factorial analysis emphasizes the strong pollution dependence of the P450-dependent enzymatic activities which vary with the season, the species, and the sex of the fish. Regression analysis of the data allowed prediction of the type of location from which the fish came (polluted or unpolluted) for 80% of the fish. This supports use of such activities as chemical pollution indicators.

In vitro inhibition of hepatic drug oxidation by thioridazine : Kinetic analysis of the inhibition of cytochrome p-450 isoform-specific reactions

The phenothiazine tranquilizer thioridazine has been associated with drug interactions in man. This study investigated the capacity of the drug to inhibit hepatic drug oxidations mediated by cytochromes P-450 (P-450) in microsomes in vitro. Thioridazine was a potent linear mixed-type inhibitor of P-450b-dependent 7-pentoxyresorufin O-depentylase activity in phenobarbital-induced rat liver. The kinetic analysis revealed the enzyme-substrate dissociation constant ( K s ) to be 1.6 μM whereas the dissociation constant of the enzyme-inhibitor complex ( K i) was 0.11 μM. In contrast, 7-ethoxyresorufin O-deethylase activity (mediated by P-450c) in β-naphthoflavone-induced rat hepatic microsomes was inhibited to a lesser extent ( K i = 2.4 μM) in relation to the K s value (0.5 μM). Spectral studies indicated that the efficiency of thioridazine binding in phenobarbital-induced microsomes was about 25-fold greater than in microsomes from β-naphthoflavone-induced rat liver. This finding is consistent with the relative capacity of thioridazine to inhibit oxidase activities catalysed by P-450b and P-450c. Mixed-function oxidase activities catalysed by other P-450s were also inhibited by thioridazine, although to a lesser extent than those catalysed by forms b and c. Thus, the 6β- and 16β-hydroxylations of androst-4-ene-3, 17-dione in hepatic microsomes from untreated rats were inhibited to a similar extent (I 50S = 52 and 43 μM, respectively). The 7α- and 16α-hydroxylase pathways were approximately only half as susceptible to inhibition by thioridazine. These findings demonstrate the capacity of thioridazine to inhibit a range of P-450-dependent drug oxidations, with those catalysed by forms b and c most susceptible. The present study strongly suggests that drug interactions elicited by thioridazine are most likely a consequence of inhibitory interactions with P-450 enzymes.

Comparison of the effects of acute and subacute treatment of phenobarbital in different strains of mice

A strain specificity has been demonstrated for the effect of subsequent administration of phenobarbital (PB), in which diethylnitrosamine (DENA)-initiated hepatocarcinogenesis was promoted in C3H mice, inhibited in B6C3F1 (C57BL × C3H) and not affected in C57BL mice. A correlation has been established between the ability of barbiturates and hydantoins to promote tumor formation and their ability to induce liver growth, hepatic DNA synthesis and mixed function oxidase activities. Therefore, we examined in these 3 strains of mice and in C3B6F1 (C3H × C57BL) mice the effect of PB administered in their drinking water for 4 days or 28 days. The liver weight to body weight ratio was increased by PB in all types of mice. Microsomal protein concentrations were increased in C57BL mice after 28 days of treatment, in C3H after both 4 days and 28 days and in B6C3F1 after 4 days of treatment. No effect upon microsomal protein content was observed in C3B6F1 mice. DNA content was increased in C3H mice, both in the 4-day and 28-day treatment groups, while the other strains showed either a decrease or no difference from control. DNA synthesis was elevated in all strains of mice after 4 days of treatment with PB, however, after 28 days of treatment there was either a much reduced increase (C57BL and C3B6F1) or no difference (C3H and B6C3F1) from controls. In all 4 types of mice after 4 and 28 days of treatment, PB increased the concentration of cytochrome P-450, the activity of aminopyrine-N-demethylase (AmDm) and 7-ethoxyresorufin-O-deethylase (ErDe) and the oxidation of testosterone (T). The oxidative metabolites of T were similar in the 4 types of mice.