Cyp1a1(-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria

To study liver toxicity and uroporphyrin (URO) accumulation and urinary excretion, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent ligand for the aryl hydrocarbon receptor (AHR), is often used as the prototype. In this study, we asked the question how important is the role of CYP1A1 in causing TCDD toxicity. Using a single large intraperitoneal dose of TCDD (200 microg/kg) and following the response over an 8-week period, we found this dose: (a) was lethal in less than 4 weeks to Cyp1a1(+/+) males but not to Cyp1a1(-/-) males or to females of either genotype; (b) caused a wasting syndrome in Cyp1a1(+/+) but not Cyp1a1(-/-) mice; (c) resulted in thymic atrophy, regardless of gender or genotype; (d) decreased spleen size and caused leukocytopenia in males but not females of either genotype; (e) caused hepatocyte hypertrophy in Cyp1a1(+/+) more so than in Cyp1a1(-/-) mice; (f) increased intrahepatocyte lipids and total liver fat content in Cyp1a1(+/+) more than Cyp1a1(-/-) males and females; and (g) caused uroporphyria in Cyp1a1(+/+) males much more than Cyp1a1(+/+) females, or in Cyp1a1(-/-) mice. Contrary to Cyp1a2(-/-) knockout mice that exhibited 15 times less accumulation of TCDD in liver than Cyp1a1/1a2(+/+) wild-type mice, Cyp1a1(-/-) mice did not show this altered TCDD distribution-indicating that CYP1A2 but not CYP1A1 is the major hepatic TCDD-binding "sink". Our data demonstrate that CYP1A1 contributes to high-dose TCDD-induced toxicity, uroporphyria, and lethality.     

Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the liver of C57BL/6J mice is a model for clinical sporadic porphyria cutanea tarda (PCT). There is massive uroporphyria, inhibition of uroporphyrinogen decarboxylase (UROD) activity, and hepatocellular damage. A variety of evidence implicates the CYP1A2 enzyme as necessary for mouse uroporphyria. Here we report that, 5 weeks after a single oral dose of TCDD (75 microg/kg), Cyp1a2(+/+) wild-type mice showed severe uroporphyria and greater than 90% decreases in UROD activity; in contrast, despite exposure to this potent agent Cyp1a2(-/-) knockout mice displayed absolutely no increases in hepatic porphyrin levels, even after prior iron overload, and no detectable inhibition of UROD activity. Plasma levels of alanine-aminotransferase (ALT) and aspartate aminotransferase (AST)-although elevated in both genotypes after TCDD exposure-were significantly less in Cyp1a2(-/-) than in Cyp1a2(+/+) mice, suggesting that the absence of CYP1A2 also affords partial protection against TCDD-induced liver toxicity. Histological examination confirmed a decrease in hepatocellular damage in TCDD-treated Cyp1a2(-/-) mice; in particular, there was no bile duct damage or proliferation that in the Cyp1a2(+/+) mice might be caused by uroporphyrin. We conclude that CYP1A2 is both necessary and essential for the potent uroporphyrinogenic effects of TCDD in mice, and that CYP1A2 also plays a role in contributing to TCDD-induced hepatocellular injury. This study has implications for both the toxicity assessment of TCDD and the hepatic injury seen in PCT patients.     

Oral exposure to benzo[a]pyrene in the mouse: detoxication by inducible cytochrome P450 is more important than metabolic activation

The cytochrome P450 (CYP1A1) enzyme metabolically activates many polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), to DNA- and protein-binding intermediates that are associated with toxicity, mutagenesis, and carcinogenesis. As a result, it is widely accepted that CYP1A1 potentiates the toxicity of this class of chemicals. In distinct contrast, we show here that CYP1A1 inducibility is essential in the detoxication of oral BaP. We compared Cyp1a1(-/-) knockout mice, having the genetic absence of the CYP1A1 enzyme, with Cyp1a1(+/+) wild-type mice. At an oral BaP dose of 125 mg/kg/day, Cyp1a1(-/-) mice died within 30 days whereas Cyp1a1(+/+) mice displayed no outward signs of toxicity. The rate of BaP clearance was 4-fold slower in Cyp1a1(-/-) than Cyp1a1(+/+) mice. The cause of death in Cyp1a1(-/-) mice receiving oral BaP seemed to be immunotoxicity, including toxic chemical depression of the bone marrow; some toxic effects in Cyp1a1(-/-) mice were noted at a BaP dose as low as 1.25 mg/kg/day. DNA post-labeling studies demonstrated dramatically higher BaP-DNA adduct levels in all Cyp1a1(-/-) tissues assayed, with the exception of the small intestine, which is probably a major site of BaP metabolism in Cyp1a1(+/+) mice. Different BaP-DNA adduct patterns were also observed between the two genotypes receiving oral BaP. Despite previous studies in vitro and in cell culture that have shown a participatory role for CYP1A1 in BaP toxicity, the present data indicate that, in the intact animal, inducible CYP1A1 is extremely important in detoxication and protection against oral BaP toxicity.     

A temporal study on the histopathological, biochemical and molecular responses of CCl(4)-induced hepatotoxicity in Cyp2e1-null mice

Previous study using Cyp2e1-null mice showed that Cyp2e1 is required in CCl(4)-induced liver injury at 24h, what remains unclear are the temporal changes in liver damage and the spectrum of genes involved in this process. We investigated the time-dependent liver changes that occurred at morphological, histopathological, biochemical and molecular levels in both Cyp2e1(+/+) and Cyp2e1(-/-) mice after treating with either corn oil or CCl(4) (1 ml/kg) for 2, 6, 12, 24 and 48 h. A pale orange colored liver, indicative of fatty infiltration, was observed in Cyp2e1(+/+) mice treated with CCl(4) for 24 and 48 h, while the Cyp2e1(+/+) mice treated with corn oil and Cyp2e1(-/-) mice treated with either corn oil or CCl(4) showed normal reddish brown colored liver. Ballooned hepatocytes with multiple vacuoles in their cytoplasm were observed in the livers of Cyp2e1(+/+) mice 24 and 48 h after treating with CCl(4). The levels of serum alanine aminotransferase and aspartate aminotransferase, markers for liver injury, were significantly higher at 12h, peaked at 24h and gradually decreased at 48 h after CCl(4) intoxication. In contrast, this kind of damage was not apparent in the Cyp2e1(-/-) mice treated with CCl(4). Altered expressions of genes related to liver cirrhosis, apoptosis, oxidative stress, xenobiotic detoxification, lipid metabolism, chemsensory signaling or tumorigenesis, structural organization, regeneration and inflammatory response were identified, and the time-dependent changes in expression of these genes were varied. Overall, the present study provides insights into the mechanism of CCl(4)-induced hepatotoxicity in animal models.     

Role of CYP3A and CYP2E1 in alcohol-mediated increases in acetaminophen hepatotoxicity: comparison of wild-type and Cyp2e1(-/-) mice.

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.     

Impact of Cyp1a2 or Ahr gene knockout in mice: implications for biomonitoring studies

Studies of the impact of phase 1 enzyme polymorphisms on genetic damage have yielded mixed results. We studied how genetic damage would be altered when specific genes were ablated under low dose conditions. METHODS: Knockouts (KO) were generated from c57bl6/J mice with mutations in Cyp1a2 or Ahr receptor that eliminated gene product function. Animals were treated topically with either 4-aminobiphenyl (4ABP) 10mg/kg, benzo(a)pyrene (BaP) 33.3mg/kg or dibenzo(c,g)carbazole (DBC) 8 mg/kg, and sacrificed after 24h. DNA from livers, skin and/or urinary bladders were isolated and (32)P-post labelled. RESULTS: Cyp1a2-/- mice did not differ in 4ABP DNA adduct levels in either urinary bladder or liver compared to wildtype. There was a sex difference in the organ affected. Cyp1a2 knockout reduced skin BAP adduct levels 50% and AHR knockout reduced skin BAP adduct levels by 90%. There was no impact of either knockout on the levels of DBC-DNA adducts in any tissue. CONCLUSIONS: Ablation of specific metabolizing enzymes had compound- and tissue-specific effects in mice. Phenotypic variability in single CYP enzymes may have minor impact in humans at low doses, but variation in the ability to induce the family of CYPs may have a greater impact.     

Role of mouse CYP2E1 in the O-hydroxylation of p-nitrophenol: comparison of activities in hepatic microsomes from Cyp2e1(-/-) and wild-type mice.

Enzymatic activities are routinely used to identify the contribution of individual forms of cytochrome P450 in a particular biotransformation. p-Nitrophenol O-hydroxylation (PNPH) has been widely used as a measure of CYP2E1 catalytic activity. However, rat and human forms of CYP3A have also been shown to catalyze this activity. In mice, the contributions of CYP3A and CYP2E1 to PNPH activity are not known. Here we used hepatic microsomes from Cyp2e1(-/-) and wild-type mice to investigate the contributions of constitutively expressed and alcohol-induced murine CYP2E1 and CYP3A to PNPH activity. In liver microsomes from untreated mice, PNPH activity was much greater in wild-type mice compared with Cyp2e1(-/-) mice, suggesting a major role for CYP2E1 in catalyzing PNPH activity. Hepatic PNPH activities were not significantly different in microsomes from male and female mice, although the microsomes from females have dramatically higher levels of CYP3A. Treatment with a combination of ethanol and isopentanol resulted in induction of CYP3A proteins in wild-type and Cyp2e1(-/-) mice, as well as CYP2E1 protein in wild-type mice. The alcohol treatment increased PNPH activities in hepatic microsomes from wild-type mice but not from Cyp2e1(-/-) mice. Our findings suggest that in untreated and alcohol-treated mice, PNPH activity may be used as a specific probe for CYP2E1 and that constitutively expressed and alcohol-induced forms of mouse CYP3A have little to no role in catalyzing PNPH activity.     

Oral benzo[a]pyrene in Cyp1 knockout mouse lines: CYP1A1 important in detoxication, CYP1B1 metabolism required for immune damage independent of total-body burden and clearance rate

CYP1A1 and CYP1B1 metabolically activate many polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene, to reactive intermediates associated with toxicity, mutagenesis, and carcinogenesis. Paradoxically, however, Cyp1a1-/- knockout mice are more sensitive to oral benzo[a]pyrene exposure, compared with wild-type Cyp1a1+/+ mice (Mol Pharmacol 65:1225, 2004). To further investigate the mechanism for this enhanced sensitivity, Cyp1a1-/-, Cyp1a2-/-, and Cyp1b1-/- single-knockout, Cyp1a1/1b1-/- and Cyp1a2/1b1-/- double-knockout, and Cyp1+/+ wild-type mice were analyzed. After administration of oral benzo[a]pyrene (125 mg/kg/day) for 18 days, Cyp1a1-/- mice showed marked wasting, immunosuppression, and bone marrow hypocellularity, whereas the other five genotypes did not. After 5 days of feeding, steady-state blood levels of benzo[a]pyrene were approximately 25 and approximately 75 times higher in Cyp1a1-/- and Cyp1a1/1b1-/- mice, respectively, than in wild-type mice. Benzo[a]pyrene-DNA adduct levels were highest in liver, spleen, and marrow of Cyp1a1-/- and Cyp1a1/1b1-/- mice. Many lines of convergent data obtained with oral benzo[a]pyrene dosing suggest that: 1) inducible CYP1A1, probably in both intestine and liver, is most important in detoxication; 2) CYP1B1 in spleen and marrow is responsible for metabolic activation of benzo[a]pyrene, which results in immune damage in the absence of CYP1A1; 3) both thymus atrophy and hepatocyte hypertrophy are independent of CYP1B1 metabolism but rather may reflect long-term activation of the aryl hydrocarbon receptor; and 4) the magnitude of immune damage in Cyp1a1-/- and Cyp1a1/1b1-/- mice is independent of plasma benzo[a]pyrene and total-body burden and clearance. Thus, a balance between tissue-specific expression of the CYP1A1 and CYP1B1 enzymes governs sensitivity of benzo[a]pyrene toxicity and, possibly, carcinogenicity.     

Short-term treatment with alcohols causes hepatic steatosis and enhances acetaminophen hepatotoxicity in Cyp2e1(-/-) mice

CYP2E1 has been reported to have an essential role in alcohol-mediated increases in hepatic steatosis and acetaminophen hepatotoxicity. We found that pretreatment of Cyp2e1(-/-) mice with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, for 7 days resulted in micro- and macrovesicular steatosis in the livers of all mice, as well as a dramatic increase in acetaminophen hepatotoxicity. In Cyp2e1(-/-) mice administered up to 600 mg acetaminophen/kg alone and euthanized 7 h later, there was no increase in serum levels of ALT. In Cyp2e1(-/-) mice pretreated with ethanol and isopentanol, subsequent exposure to 400 or 600 mg acetaminophen/kg resulted in centrilobular necrosis in all mice with maximal elevation in serum levels of ALT. Acetaminophen-mediated liver damage was similar in males and females. Hepatic microsomal levels of APAP activation in untreated females were similar to those in males treated with the alcohols. However, the females, like the males, required pretreatment with the alcohols in order to increase APAP hepatotoxicity. These findings suggest that, in the Cyp2e1(-/-) mice, the alcohol-mediated increase in acetaminophen hepatotoxicity involves the contribution of other factors, in addition to induction of CYP(s) that activate acetaminophen. Alternatively, CYP-mediated activation of acetaminophen measured in vitro may not reflect the actual activity in vivo. Our findings that a 7-day treatment with ethanol and isopentanol causes extensive hepatic steatosis and increases acetaminophen hepatotoxicity in Cyp2e(-/-) mice indicate that CYP2E1 is not essential for either response.     

Role of P450 1A1 and P450 1A2 in bioactivation versus detoxication of the renal carcinogen aristolochic acid I: studies in Cyp1a1-/-, Cyp1a2-/-, and Cyp1a1/1a2-/- mice

Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.     

A novel defensive mechanism against acetaminophen toxicity in the mouse lateral nasal gland: role of CYP2A5-mediated regulation of testosterone homeostasis and salivary androgen-binding protein expression

To identify novel factors or mechanisms that are important for the resistance of tissues to chemical toxicity, we have determined the mechanisms underlying the previously observed increases in resistance to acetaminophen (APAP) toxicity in the lateral nasal gland (LNG) of the male Cyp2g1-null/Cyp2a5-low mouse. Initial studies established that Cyp2a5-null mice, but not a newly generated strain of Cyp2g1-null mice, were resistant to APAP toxicity in the LNG; therefore, subsequent studies were focused on the Cyp2a5-null mice. Compared with the wild-type (WT) male mouse, the Cyp2a5-null male mouse had intact capability to metabolize APAP to reactive intermediates in the LNG, as well as unaltered circulating levels of APAP, APAP-GSH, APAP-glucuronide, and APAP-sulfate. However, it displayed reduced tissue levels of APAP and APAP-GSH and increased tissue levels of testosterone and salivary androgen-binding protein (ABP) in the LNG. Furthermore, we found that ABP was able to compete with GSH and cellular proteins for adduction with reactive metabolites of APAP in vitro. The amounts of APAP-ABP adducts formed in vivo were greater, whereas the amounts of APAP adducts formed with other cellular proteins were substantially lower, in the LNG of APAP-treated male Cyp2a5-null mice compared with the LNG of APAP-treated male WT mice. We propose that through its critical role in testosterone metabolism, CYP2A5 regulates 1) the bioavailability of APAP and APAP-GSH (presumably through modulation of the rates of xenobiotic excretion from the LNG) and 2) the expression of ABP, which can quench reactive APAP metabolites and thereby spare critical cellular proteins from inactivation.     

Role of CYP1A2 and CYP2E1 in the pentoxifylline ciprofloxacin drug interaction

In this study the drug interaction between ciprofloxacin (CIPRO) and pentoxifylline (PTX) was investigated and the role of CYP1A2 in the drug interaction was determined with the aid of a selective CYP1A2 inhibitor, furafylline (FURA), and the Cyp1A2 knockout mouse. Serum concentrations of PTX (83.4+/-1 micromol/l) and metabolite-1 (M-1) (13.7+/-2.8 micromol/l) following a single injection of PTX (100 mg/kg i.p.) were significantly higher (P<0.05) in mice treated with CIPRO (25 mg/kg i.p. 9 days) compared to serum concentrations of PTX (46.3+/-0.5 micromol/l) and M-1 (6.4+/-1.1 micromol/l) in mice administered saline. Murine hepatic microsomes were incubated with PTX alone or the combination of PTX and CIPRO. The metabolism of PTX in the murine hepatic microsomes containing both CIPRO and PTX was significantly decreased compared to microsomes incubated with PTX alone, suggesting that CIPRO may inhibit the metabolism of PTX. To further clarify the role of CYP1A2 in the metabolism of PTX in mice, the effect of a selective CYP1A2 mechanism based inhibitor, FURA, on the metabolism of PTX was investigated and our results indicate that FURA inhibited metabolism of PTX. We then investigated PTX elimination in the Cyp1A2 knockout mouse. Blood levels of PTX were assessed at 2 and 20 min following a single injection of PTX (32 mg/kg i.v). Serum concentration of PTX was determined in Cyp1A2 knockout mice compared to Cyp1A2 wild type control mice. The serum concentration of PTX in Cyp1A2 wild type mice (n=9) was 22.2+/-3.2 micromol/l at 20 min following injection of PTX. The serum concentration of PTX in Cyp1A2 knockout mice (n=11) was significantly elevated at 20 min following injection of PTX compared to Cyp1A2 wild type mice. These results clearly indicate that inhibition of CYP1A2 catalytic activity that occurs in the Cyp1A2 knockout mice is sufficient to alter metabolism of PTX and result in markedly elevated levels in serum of Cyp1A2 knockout mice. The results of Western analysis in murine microsomes suggest that CYP1A2 protein levels were not altered by CIPRO indicating that CIPRO did not downregulate Cyp1A2. The results of Western analysis also indicated that CIPRO treatment increased CYP2E1 in mouse microsomes and the implications of these will be discussed.     

Role of CYP2E1 in thioacetamide-induced mouse hepatotoxicity

Previous experiments showed that treatment of mice and rats with thioacetamide (TAA) induced liver cell damage, fibrosis and/or cirrhosis, associated with increased oxidative stress and activation of hepatic stellate cells. Some experiments suggest that CYP2E1 may be involved in the metabolic activation of TAA. However, there is no direct evidence on the role of CYP2E1 in TAA-mediated hepatotoxicity. To clarify this, TAA-induced hepatotoxicity was investigated using Cyp2e1-null mice. Male wild-type and Cyp2e1-null mice were treated with TAA (200 mg/kg of body weight, single, i.p.) at 6 weeks of age, and hepatotoxicity examined 24 and 48 h after TAA treatment. Relative liver weights of Cyp2e1-null mice were significantly different at 24 h compared to wild-type mice (p<0.01). Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in Cyp2e1-null mice were significantly different at both time points compared to wild-type mice (p<0.01). Histopathological examination showed Cyp2e1-null mice represented no hepatototoxic lesions, in clear contrast to severe centriobular necrosis, inflammation and hemorrhage at both time points in wild-type mice. Marked lipid peroxidation was also only limited to wild-type mice (p<0.01). Similarly, TNF-alpha, IL-6 and glutathione peroxidase mRNA expression in Cyp2e1-null mice did not significantly differ from the control levels, contrasting with the marked alteration in wild-type mice (p<0.01). Western blot analysis further revealed no increase in iNOS expression in Cyp2e1-null mice. These results reveal that CYP2E1 mediates TAA-induced hepatotoxicity in wild-type mice as a result of increased oxidative stress.     

Quantitative analysis of benzo[a]pyrene biotransformation and adduct formation in Ahr knockout mice

Benzo[a]pyrene (BP) is an ubiquitous environmental pollutant with potent mutagenic and carcinogenic properties. The Ah receptor (Ahr) is involved in the metabolic activation of BP and is therefore important in the induction of chemical carcinogenesis. In this study, the relationship between Ahr genotype and biotransformation of BP in internal organs was investigated in Ahr (+/+), Ahr (+/-) and Ahr (-/-) mice. The mice were treated with BP (100mg/kg) by gavage. Gene expression was measured after 24h by real-time RT-PCR and showed induction of Cyp1a1 in liver and lung, and Cyp1b1 in lung in both Ahr (+/+) and Ahr (+/-). No induction of the Cyp genes was observed in the Ahr (-/-). There was a significant basal expression of Cyp1b1 in the liver of all genotypes, and this expression was independent of the BP exposure. Analyzed by HPLC-fluorescence, there were increased levels of protein and DNA adducts, metabolites, conjugates and unmetabolized BP in the internal organs of Ahr (-/-) as compared to Ahr (+/+) and Ahr (+/-) mice. This may be partly explained by a delayed bioactivation of BP in the Ahr deficient mice. The BP metabolism observed in the Ahr (-/-) mice is also evidence of an Ahr independent biotransformation of BP.     

Mdr1 limits CYP3A metabolism in vivo

We determined whether the drug efflux protein P-glycoprotein (Pgp) could influence the extent of CYP3A-mediated metabolism of erythromycin, a widely used model substrate for CYP3A. We compared CYP3A metabolism of erythromycin (a Pgp substrate) using the erythromycin breath test in mice proficient and deficient of mdr1 drug transporters. We first injected mdr1(+/+) mice with [(14)C]N-methyl erythromycin and measured the rate of appearance of (14)CO(2) in the breath as a measure of hepatic CYP3A activity. Animals treated with CYP3A inducers or inhibitor showed accelerated or diminished (14)CO(2) in the breath, respectively. The erythromycin breath test was next administered to mdr1a(-/-) and mdr1a/1b(+/+) and (-/-) mice. These animals had equivalent levels of immunoreactive CYP3A and CYP3A activity as measured by erythromycin N-demethylase activity in liver microsomes. Nevertheless, the rate of (14)CO(2) appearance in the breath showed no relationship with these measurements of CYP3A, but changed proportionally to expression of mdr1. The average breath test (14)CO(2) area under the curves were 1.9- and 1.5-fold greater in mdr1a/1b(-/-) and mdr1a(-/-) mice, respectively, compared with (+/+) mice, and CER(max) was 2-fold greater in mdr1a/1b(-/-) compared with (+/+) mice. We conclude that Pgp, by limiting intracellular substrate availability can be an important determinant of CYP3A metabolism of numerous medications that are substrates for CYP3A and Pgp.     

Effects of benzo[a]pyrene,aromatic amines,and a combination of both on CYP1A1 activities in RT-4 human bladder papilloma cells

ABSTRACT

The interaction of arylamines and polycyclic aromatic hydrocarbons (PAH) is of particular interest in the etiology of bladder cancer. The aim of this study was to (1) examine the metabolic capacity of RT-4 human bladder papilloma cells and (2) investigate the influence of aromatic amines on the induction of cytochrome P-450 1A1 (CYP1A1) activity and their effects on benzo[a]pyrene (BaP)-induced CYP1A1 activities. Cells were incubated for 24 h with different concentrations of BaP, 1- or 2-naphthylamine (NA), 2-, 3-, or 4-aminobiphenyl (ABP), and binary mixtures consisting of 1 µM BaP and different concentrations of each arylamine. Changes in CYP1A1 activities were measured at concentrations with no or only low cytotoxicity and accompanied by specific protein detection. Several phase I and II enzymes relevant to metabolism of PAH and arylamines were present in RT-4 cells. Concentration-dependent elevation in CYP1A1 activities accompanied by increasing protein levels was found after treating cells with BaP and 1- or 2-NA. The majority of synergistic effects in binary mixtures were less than additive. In contrast, concentration-dependent inhibition was observed for 2-, 3-, and 4-ABP and in both the presence and absence of BaP. Our results suggest that RT-4 cells represent a reliable model cell line to study arylamine- and PAH-induced effects in vitro and that BaP-induced CYP1A1 activities are modulated by aromatic amines. In general, the direction of the effect depends upon the aromatic amine, rather than being unidirectional for aromatic amines.     

Functional Cyp2e1 is required for substantial in vivo formation of 2,5-hexanedione from n-hexane in the mouse

Neurotoxicity of n-hexane is mediated by its metabolite 2,5-hexanedione (2,5-HD). Cytochrome P4502E1 (CYP2E1) has been suggested but not shown to be involved in the formation of the metabolite. An objective of the current study was to assess the essentiality of CYP2E1 for in vivo 2,5-HD formation from n-hexane. This was accomplished by comparing urinary levels of the gamma-diketone in n-hexane-treated mice in which the Cyp2e1 gene has been deleted (Cyp2e1-/-) with that in n-hexane-treated wild-type (Cyp2e1+/+) mice. 2,5-HD was detectable not as the free compound but as further metabolites, at levels that were comparable in both strains of mice, following a daily 200 mg/kg i.p. dose of the alkane for 10 days. Continued daily n-hexane treatment resulted in increased urinary levels of 2,5-HD metabolites in Cyp2e1+/+ but not in Cyp2e1-/- mice. Only in Cyp2e1+/+ mice and only on day 21 of n-hexane treatment was a trace level of unchanged 2,5-HD detected. 3-Hexanol was the only other n-hexane metabolite detected in the mice but its concentration was higher in Cyp2e1-/- than in Cyp2e1+/+ mice. In n-hexane-treated rats, in contrast to mice, multiple metabolites of the alkane, including unchanged 2,5-HD, were detected. The results indicate that substantial in vivo formation of 2,5-HD from n-hexane in the mouse requires CYP2E1, and suggest that further detoxification of the metabolite may be very efficient in this species.