ß-Naphthoflavone treatment attenuates neonatal hyperoxic lung injury in wild type and Cyp1a2-knockout mice.

Exposure to supraphysiological concentrations of oxygen (hyperoxia) leads to bronchopulmonary dysplasia (BPD), one of the most common pulmonary morbidities in preterm neonates, which is more prevalent in males than females. Beta-naphthoflavone (BNF) is protective against hyperoxic lung injury in adult and neonatal wild type (WT) mice and in and mice lacking Cyp1a1gene. In this investigation, we tested the hypothesis that BNF treatment will attenuate neonatal hyperoxic lung injury in WT and Cyp1a2-/- mice, and elucidated the effect of sex-specific differences. Newborn WT or Cyp1a2-/- mice were treated with BNF (10mg/kg) or the vehicle corn oil (CO) i.p., from postnatal day (PND) 2 to 8 once every other day, while being maintained in room air or hyperoxia (85% O2) for 14days. Hyperoxia exposure lead to alveolar simplification and arrest in angiogenesis in WT as well as Cyp1a2-/- mice No significant differences were seen between WT and Cyp1a2-/- mice. Cyp1a2-/- female mice had better preservation of pulmonary angiogenesis at PND15 compared to similarly exposed males. BNF treatment attenuated lung injury and inflammation in both genotypes, and this was accompanied by a significant induction of hepatic and pulmonary CYP1A1 in WT but not in Cyp1a2-/- mice. BNF treatment increased NADPH quinone oxidoreductase (NQO1) mRNA levels in Cyp1a2-/- mouse livers compared to WT mice. These results suggest that BNF is protective in neonatal mice exposed to hyperoxia independent of CYP1A2 and this may entail the protective effect of phase II enzymes like NQO1.

Smoke carcinogens cause bone loss through the aryl hydrocarbon receptor and induction of Cyp1 enzymes.

Smoking is a major risk factor for osteoporosis and fracture, but the mechanism through which smoke causes bone loss remains unclear. Here, we show that the smoke toxins benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interact with the aryl hydrocarbon receptor (Ahr) to induce osteoclastic bone resorption through the activation of cytochrome P450 1a/1b (Cyp1) enzymes. BaP and TCDD enhanced osteoclast formation in bone marrow cell cultures and gavage with BaP stimulated bone resorption and osteoclastogenesis in vivo. The osteoclastogenesis triggered by BaP or RANK-L was reduced in Ahr(-/-) cells, consistent with the high bone mass noted in Ahr(-/-) male mice. The receptor activator of NF-κB ligand (RANK-L) also failed to induce the expression of Cyp1 enzymes in Ahr(-/-) cells. Furthermore, the osteoclastogenesis induced by TCDD was lower in Cyp1a1/1a2(-/-) and Cyp1a1/1a2/1b1(-/-) cultures, indicating that Ahr was upstream of the Cyp enzymes. Likewise, the pharmacological inhibition of the Cyp1 enzymes with tetramethylsilane or proadifen reduced osteoclastogenesis. Finally, deletion of the Cyp1a1, Cyp1a2, and Cyp1b1 in triple knockout mice resulted in reduced bone resorption and recapitulated the high bone mass phenotype of Ahr(-/-) mice. Overall, the data identify the Ahr and Cyp1 enzymes not only in the pathophysiology of smoke-induced osteoporosis, but also as potential targets for selective modulation by new therapeutics.

Phenacetin pharmacokinetics in CYP1A2-deficient beagle dogs.

Phenacetin is widely used as an in vitro probe to measure CYP1A2 activity across species. To investigate whether phenacetin can be used as an in vivo probe substrate to phenotype CYP1A2 activity in dogs, beagle dogs previously genotyped for a single nucleotide polymorphism that yields an inactive CYP1A2 protein were selected and placed into one of three groups: CC (wild-type), CT (heterozygous), or TT (homozygous mutants). The dogs were dosed with phenacetin orally at 5 and 15 mg/kg and intravenously at 15 mg/kg. Plasma samples were analyzed by liquid chromatography-tandem mass spectrometry, and phenacetin and its primary metabolite, acetaminophen, were monitored. After intravenous dosing, all groups showed similar exposure of phenacetin irrespective of genotype. After oral dosing at 15 mg/kg, the exposure of phenacetin in CC and CT dogs was similar, but phenacetin exposure was 2-fold greater in TT dogs. Exposure of the metabolite, acetaminophen, was similar in all groups; however, the mean acetaminophen/phenacetin ratio in TT dogs was 1.7 times less than that observed in CC dogs. Similar trends between the groups of dogs with respect to phenacetin exposure were also observed after a lower 5 mg/kg p.o. dose of phenacetin; however, a proportionally greater amount of acetaminophen was generated. Although oral exposure of phenacetin was 2-fold higher and acetaminophen exposure was 2-fold lower in CYP1A2-deficient (TT) dogs, these results were considered modest and suggest that phenacetin is not a selective or robust in vivo probe to measure CYP1A2 enzyme activity in the dog.

NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I.

Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)-the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1⁻/⁻, Cyp1a2⁻/⁻ and Cyp1a1/1a2⁻/⁻ knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Higher susceptibility to aflatoxin B(1)-related hepatocellular carcinoma in glycine N-methyltransferase knockout mice.

In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N-methyltransferase (GNMT) interacts with aflatoxin B(1) (AFB(1)) and reduces both AFB(1)-DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt-/-) mice developed HCC, with first dysplastic hepatocellular nodules detected at mean ages of 17 and 16.5 months, respectively. In our study, we tested our hypothesis that male and female Gnmt-/- mice are susceptible to AFB(1) carcinogenesis, and that the absence of Gnmt expression may accelerate AFB(1)-induced liver tumorigenesis. We inoculated Gnmt-/- and wild-type mice intraperitoneally with AFB(1) at 7 days and 9 weeks of age and periodically examined them using ultrasound. Dysplastic hepatocellular nodules were detected in six of eight males and five of five females at 12.7 and 12 months of ages, respectively. Dysplastic hepatocellular nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt-/- mice were diagnosed as having HCC, ∼6 months earlier than AFB(1)-treated wild-type mice. Results from microarray and real-time PCR analyses indicate that five detoxification pathway-related genes were downregulated in AFB(1)-treated Gnmt-/- mice: Cyp1a2, Cyp3a44, Cyp2d22, Gsta4 and Abca8a. In summary, we observed overall higher susceptibility to AFB(1)-related HCC in Gnmt-/- mice, further evidence that GNMT overexpression is an important contributing factor to liver cancer resistance.

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.

EMQN best practice guidelines for molecular genetic testing and reporting of 21-hydroxylase deficiency.

Molecular genetic testing for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is offered worldwide and is of importance for differential diagnosis, carrier detection and adequate genetic counseling, particularly for family planning. In 2008 the European Molecular Genetics Quality Network (EMQN) for the first time offered a European-wide external quality assessment scheme for CAH (due to 21-OH deficiency). The interest was great and over the last years at about 60 laboratories from Europe, USA and Australia regularly participated in that scheme. These best practice guidelines were drafted on the basis of the extensive knowledge and experience got from those annually organized CAH-schemes. In order to obtain the widest possible consultation with practicing laboratories the draft was therefore circulated twice by EMQN to all laboratories participating in the EQA-scheme for CAH genotyping and was updated by that input. The present guidelines address quality requirements for diagnostic molecular genetic laboratories, as well as criteria for CYP21A2 genotyping (including carrier-testing and prenatal diagnosis). A key aspect of that article is the use of appropriate methodologies (e.g., sequencing methods, MLPA (multiplex ligation dependent probe amplification), mutation specific assays) and respective limitations and analytical accuracy. Moreover, these guidelines focus on classification of variants, and the interpretation and standardization of the reporting of CYP21A2 genotyping results. In addition, the article provides a comprehensive list of common as well as so far unreported CYP21A2-variants.

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

UNLABELLED: 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.

Sex-specific differences in hyperoxic lung injury in mice: role of cytochrome P450 (CYP)1A.

Sex-specific differences in pulmonary morbidity in adults and preterm infants are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. Cytochrome P450 (CYP) 1A enzymes have been shown to play a mechanistic role in hyperoxic lung injury (HLI) in animal models. Whether CYP1A enzymes contribute to gender-specific differences in relation to HLI is unknown. In this investigation, we tested the hypothesis that mice will display gender-specific differences in HLI, and that this phenomenon will be altered in mice lacking the genes for Cyp1a1 or 1a2. Eight week-old male and female wild type (WT) (C57BL/6J) mice, Cyp1a1-/-, and Cyp1a2-/- mice were exposed to 72h of hyperoxia (FiO2>0.95). Lung injury and inflammation were assessed and pulmonary and hepatic CYP1A1 and CYP1A2 levels were quantified at the enzyme activity, protein and mRNA level. Upon exposure to hyperoxia, liver and lung microsomal proteins showed higher pulmonary CYP1A1 (apoprotein level and activity) in WT females compared to WT males and a greater induction in hepatic CYP1A2 mRNA levels and activity in WT females after hyperoxia exposure. The gender based female advantage was lost or reversed in Cyp1a1-/- and Cyp1a2-/- mice. These findings suggest an important role for CYP1A enzymes in the gender-specific modulation of hyperoxic lung injury.

Role of cytochrome P450 1A2 in bilirubin degradation Studies in Cyp1a2 (-/-) mutant mice.

In congenital jaundice, which is due to defects of bilirubin gluruconidation, bilirubin is degraded by an alternative pathway into unidentified products. Previously, it was shown that plasma bilirubin levels can be decreased in rats with this defect by inducers of CYP1A enzymes. Here, liver microsomes from rats or mice treated with beta-naphthoflavone (BNF) or 3-methylcholanthrene (3 MC) had increased activity for bilirubin degradation. The activity was further stimulated by addition of the coplanar molecule 3,4,3',4'-tetrachlorobiphenyl (TCB). There was more stimulation of bilirubin degradation by TCB in microsomes from BNF-treated rats than in microsomes from BNF-treated mice. CYP1A1 to CYP1A2 ratios were greater in rats treated with BNF. In Cyp1a2 (-/-) mutant mice, 3-MC treatment did not increase the rate of bilirubin degradation, but TCB increased this degradation severalfold. Between SWR and C57BL/6 inbred mouse strains that have a 2-fold difference in hepatic constitutive CYP1A2 levels, there was also a 2-fold difference in bilirubin degradation; TCB did not stimulate in either strain. We conclude that CYP1A2 is responsible for microsomal bilirubin degradation in the absence of TCB. TCB was required for bilirubin degradation by CYP1A1. Manipulation of CYP1A2 may be of therapeutic benefit in patients with these diseases of bilirubin conjugation.

Role of CYP2A5 and 2G1 in acetaminophen metabolism and toxicity in the olfactory mucosa of the Cyp1a2(-/-) mouse.

Acetaminophen (AP) is a widely-used analgesic agent that has been linked to human liver and kidney disease with prolonged or high-dose usage. In rodents, the target organs that are affected include liver, kidney, and the olfactory mucosa. AP toxicity requires cytochrome P450(CYP)-mediated metabolic activation, and the isozymes CYP1A2, 2E1, and 3A are known to activate AP in the human. In the present study, we determined that olfactory mucosal toxicity of AP was not different between the Cyp1a2(+/+) wild-type and the Cyp1a2(-/-) knockout mouse, whereas the hepatic toxicity of AP was significantly diminished in Cyp1a2(-/-) mice. Western blots of olfactory mucosa revealed that CYP2E1 and CYP3A levels are similar between untreated Cyp1a2(+/+) and Cyp1a2(-/-) mice. Diallyl sulfide (DAS), a known inhibitor of CYP2E1 and of CYP2A10/2A11 (the rabbit orthologue of mouse CYP2A5), completely eliminated olfactory toxicity of AP in both the Cyp1a2(-/-) and wild-type mouse olfactory mucosa. We found that heterologously expressed mouse CYP2A5 and CYP2G1 enzymes (known to be present in olfactory mucosa) form 3-hydroxyacetaminophen (3-OH-AP) and 3-(glutathion-S-yl)acetaminophen (GS-AP); CYP2A5 is considerably more active than 2G1. Addition of GSH caused increases in GS-AP proportional to decreases in 3-OH-AP, suggesting that these two metabolites arise from a common precursor or are formed by way of competing pathways. We also found that both CYP2A5 and CYP2G1 are inhibitable by DAS in vitro. These studies provide strong evidence that, in addition to CYP2E1, CYP2A5 and 2G1 are important in AP bioactivation in the mouse olfactory mucosa and that CYP1A2 appears to be of minor importance for AP olfactory toxicity.

CYP1A2 is not required for 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced immunosuppression.

One of the most sensitive and reproducible immunotoxic endpoints of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure is suppression of the antibody response to sheep red blood cells (SRBCs) in mice. Immunosuppression occurs in concert with hepatomegaly and associated induction of several hepatic cytochrome P450 enzymes, including CYP1A2 which is responsible for the hepatic sequestration of TCDD. In this study, TCDD-induced immunosuppression was evaluated in C57BL/6N CYP1A2 (+/+) wild-type and compared with that of age-matched CYP1A2 (-/-) knockout and CYP1A2 (+/-) heterozygous female mice. Groups of mice were given a single gavage dose of 0, 0.03, 0.1, 0.3, 1.0, 3.0 or 10.0microg TCDD/kg, followed 7 days later by immunization with SRBCs. Serum was obtained 5 days after immunization and body, spleen, thymus and liver weights were measured. sheep red blood cell (SRBC) antibody titers were determined by an enzyme-linked immunosorbent assay (ELISA). Anti-SRBC titers were suppressed at 1.0, 1.0 and 0.3microg TCDD/kg for CYP1A2 (+/+), CYP1A2 (+/-), and CYP1A2 (-/-) mice, respectively, which indicated a three-fold increase in TCDD-induced immunosuppression for the CYP1A2 (-/-) mice. This increase in TCDD-induced immunosuppression may be due to the inability of CYP1A2 (-/-) mice to sequester TCDD in the liver leading to a higher dose to the immune system. In CYP1A2 (+/+) mice, a dose of 3.0microg TCDD/kg was sufficient to increase the liver weight, while in CYP1A2 (-/-) mice no increase in liver weight was observed. Application of analysis of variance and dose-response modeling approaches indicate that there is little evidence that the immunosuppression dose-response curves, for the three strains, differ in the lower part of the dose-response range. Thus, CYP1A2 is not required for TCDD-induced immunosuppression in the mouse.

Effect of CYP1A2 deficiency on heterocyclic amine DNA adduct levels in mice.

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20-30% of the levels in WT mice, a difference that was statistically significant (Student's t-test, P < 0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6+/-0.6 vs 4.6+/-0.7, respectively, relative adduct labeling (RAL) x 10(8), mean+/-S.E.M., n = 3-5 mice). In the mammary gland, however, there was no difference in IQ-DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP-DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP-DNA adducts in liver and kidney of WT mice were 9.9+/-1.1 and 22.5+/-6.9, respectively, whereas no PhIP-DNA adducts were detected in either organ of KO mice (limit of detection, 1.4-2.8 x 10(9)). PhIP-DNA adduct levels in mammary gland and colon of WT mice were 47.1+/-9.5 and 58.0+/-21.7, respectively, but accordingly only 3.8+/-0.7 and 5.4+/-0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.

Ahrd Cyp1a2(-/-) mice show increased susceptibility to PCB-induced developmental neurotoxicity.

Polychlorinated biphenyls (PCBs) are developmental neurotoxicants that produce cognitive and behavioral changes in children exposed during gestation and lactation. Coplanar PCBs bind the aryl hydrocarbon receptor (AHR) and can be sequestered in liver by cytochrome P450 1A2 (CYP1A2). The AHR is a ligand-activated transcription factor which increases expression of the CYP1 family, including CYP1A2. Our previous work examining genetic susceptibility to developmental PCB neurotoxicity showed that Ahr(b)Cyp1a2(-/-) mice with the high-affinity Ahr(b) allele and lacking CYP1A2 were most susceptible while Ahr(b)Cyp1a2(+/+) and poor-affinity Ahr(d)Cyp1a2(+/+) mice were resistant. To follow up, a fourth line of mice was generated with the Ahr(d)Cyp1a2(-/-) genotype and compared with the background strain Ahr(b)Cyp1a2(+/+). Dams received a PCB mixture or the corn oil vehicle at gestational Day 10 (GD10) and postnatal Day 5 (PND5). Offspring were tested at PND60 in open field locomotor, acoustic startle with pre-pulse inhibition (PPI), novel object recognition and Morris water maze. Locomotor activity was increased in PCB-treated Ahr(b)Cyp1a2(+/+) mice, but no differences were seen in control vs. PCB-treated Ahr(d)Cyp1a2(-/-) mice. PCB-treated Ahr(d)Cyp1a2(-/-) mice had a higher baseline startle response and significantly reduced pre-pulse inhibition at the 74dB level compared with corn oil-treated controls (P<0.05). PCB-treated Ahr(d)Cyp1a2(-/-) mice had impairments in novel objective recognition (P<0.05) and during all three hidden platform phases of Morris water maze (P<0.01). Combined with our previous findings, these results indicate Cyp1a2 genotype is more important in susceptibility to PCB-induced deficits in learning and memory, but Ahr genotype appears more important when assessing acoustic startle-PPI and locomotor activity.

Metabolic activation of phenanthrene by human and mouse cytochromes P450 and pharmacokinetics in CYP1A2 knockout mice.

In the present study V79 Chinese hamster cells were genetically engineered for stable expression of the cytochromes P450 1A1, 1A2, 1B1, and 2E1 from man and mouse to investigate species-specific differences in the regioselective metabolism and toxicity of phenanthrene (Phe), the simplest polycyclic aromatic hydrocarbon (PAH) forming a bay-region. Phe is present in various environmental samples and serves as a model substrate for PAH exposure in human biomonitoring studies. For this reason we explored metabolite profiles and metabolite-dependent cytotoxic activities in vitro. The total turnover of CYP-mediated transformation of Phe was as follows: human CYP1B1>CYP1A1>CYP1A2>>CYP2E1, and for mouse CYP1A2>>CYP2E1>CYP1A1. Striking species differences were seen as mouse CYP1B1 did not activate Phe at all, but human CYP1B1 exhibited a significant metabolic turnover comparable to CYP1A1 and CYP1A2. In vivo studies monitoring the whole blood Phe elimination in CYP1A2 knockout and wild-type mice after oral administration confirmed involvement of CYP1A2 in the bioactivation of Phe, but other processes must contribute also. Our data suggest that in humans not only CYP1A2 expressed solely in the liver plays a crucial role in Phe metabolism, but also constitutively expressed extrahepatic CYP1B1 in tissues such as lung, kidney or intestine. This finding will substantially improve the validity of human biomonitoring studies using individual Phe metabolites for the assessment of PAH exposure.

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line.

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.

Genetic polymorphism of cytochrome P450s in beagles: possible influence of CYP1A2 deficiency on toxicological evaluations.

A number of human cytochrome P450 (CYP) isozymes have been shown to be genetically polymorphic, and extensive pharmaceutical studies have been conducted to characterize the clinical relevance of the polymorphism. Although the beagle is extensively used in the safety assessment studies of new drug candidates and agricultural chemicals, only a limited number of studies have been reported on the significance of the CYP isozyme polymorphism in dogs. Recently, a single nucleotide polymorphism that results in a deficiency of canine CYP1A2 was discovered. This deficiency was shown to significantly alter the pharmacokinetic behavior of two drugs, and can be associated with a large inter-individual difference in the kinetic behavior of a third. In this article, the five genetically polymorphic canine CYP isozymes that have been reported so far are reviewed, and the altered pharmacokinetics of the drugs concerned are described. Although little information on toxicological relevance has been reported, it is possible that the modified pharmacokinetics may also cause altered toxic responses as well. This phenomenon may occur only with the types of chemicals that are eliminated mainly through polymorphic-enzyme mediated metabolism. However, it is recommended that genetically pure beagles are used for the toxicity studies and safety assessment of new chemical entities in order to reduce the potential inter-individual differences.

The canine CYP1A2 deficiency polymorphism dramatically affects the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-D]-pyrimidine-2-(1H)-one (YM-64227), a phosphodiesterase type 4 inhibitor.

In a previous study, it was shown that the novel canine single nucleotide polymorphism (SNP) CYP1A2 1117C>T yields an inactive enzyme. In this study, the effect that this SNP has on the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one (YM-64227) was investigated. Plasma concentrations of the unchanged drug and five of its metabolites (MM-1 to MM-5) were determined after either intravenous or oral administration of YM-64227 to genotyped dogs (C/C, C/T, and T/T groups). Liver microsomes were prepared from these dogs to determine the in vitro metabolic clearance of YM-64227. After a single oral administration, the maximum plasma concentration and absolute bioavailability of YM-64227 in the T/T group were 17.1 times and 27.2 times higher than those in the C/C group, respectively, whereas the pharmacokinetics of YM-64227 after intravenous administration were not affected by genotype. The metabolic profiles in the T/T group were quite distinct from the others; i.e., the main metabolite was MM-2 in the C/C group, whereas MM-1 and MM-5 were the main metabolites in the T/T group. The formation clearances of MM-2 and MM-3 in the microsomes derived from T/T type dogs were significantly lower, whereas those of MM-1, MM-4, and MM-5 were not affected. A statistically significant correlation was observed between the in vivo and in vitro metabolic intrinsic clearances (r = 0.82, p < 0.001). The canine CYP1A2 1117C>T SNP proved to be responsible for a substantial portion of the interindividual variability in the pharmacokinetics of YM-64227.

Elucidation of the effects of the CYP1A2 deficiency polymorphism in the metabolism of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1h)-one (YM-64227), a phosphodiesterase type 4 inhibitor, and its metabolites in dogs.

The canine CYP1A2 1117 C>T single nucleotide polymorphism is responsible for a substantial portion of the interindividual variability seen in the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one (YM-64227). The purpose of this study is to investigate the contribution of CYP1A2 to the metabolism of YM-64227 and its five metabolites (MM-1 to MM-5), as well as to determine the interindividual variability between the pharmacokinetic profiles of the compounds with respect to the CYP1A2 deficiency polymorphism. alpha-Naphthoflavone and anti-CYP1A1/2 antibody inhibited the metabolic activities at which MM-2 and MM-3 were formed from YM-64227 in C/C- and C/T-type microsomes. In T/T type, the rate of MM-2 and MM-3 formation was lower, and alpha-naphthoflavone and anti-CYP1A1/2 antibody were shown to have no effect. A positive correlation between the overall metabolism of YM-64227 and phenacetin O-deethylation, a CYP1A2 activity marker, was observed in all the genotypes. The in vitro metabolic clearances in the T/T type of MM-1, MM-3, MM-4, and MM-5 were less than 50% lower than those in the C/C type. The anti-CYP1A1/2 antibody inhibited the metabolism of MM-1, MM-3, MM-4, and MM-5 in the C/C and C/T types. These results suggest that the formation of MM-2 and MM-3 from YM-64227 is catalyzed by CYP1A2, and that CYP1A2 contributes mainly to the subsequent metabolism of the primary metabolites of YM-64227, with the exception of MM-2. It is possible that the interindividual variability of YM-64227 with respect to the CYP1A2 deficiency polymorphism is caused by a decrease in the metabolic activities of both the unchanged drug and its metabolites.

Role of CYP1A2 in the hepatotoxicity of acetaminophen: investigations using Cyp1a2 null mice.

Acetaminophen (APAP) is known to cause centrilobular hepatic necrosis under overdose conditions. This is thought to be mediated via the P450-generated reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI). Initially, NAPQI is detoxified by conjugation with glutathione (GSH), but once GSH is depleted, NAPQI reacts more extensively with hepatic proteins leading to hepatocellular damage. The P450 isoforms thought to be responsible for APAP hepatotoxicity in humans are CYP2E1, CYP1A2, and CYP3A4, and thus, we have investigated the effect of murine Cyp1a2 on APAP hepatotoxicity using Cyp1a2 knockout mice (Liang et al., Proc. Natl. Acad. Sci. USA 93, 1671-1676, 1996). Doses of 250 mg/kg were markedly hepatotoxic in these mice, and surprisingly, deaths only occurred in the knock-out and heterozygote mice over a 24-h period after dosing. Furthermore, there were no significant differences among survivors of any genotype in serum ALT concentrations, a well correlated indicator of APAP hepatotoxicity in mice. Finally, no differences were observed in the urinary metabolites excreted ove the 24-h period, including those derived from GSH conjugation of the major reactive metabolite NAPQI. Consistent with the effects on hepatotoxicity and metabolism, 2 h after hepatotoxic doses (500 mg/kg, i.p.) of APAP no significant differences were observed in total whole liver homogenate nonprotein thiol concentrations among the three genotypes even though hepatic thiols were decreased compared to control animals (> 90%). In addition, when the liver cytosol and microsome samples were examined by immunoblotting for the presence of APAP-protein adducts using a specific antiserum, there were no observable differences in either the intensity of staining or in the spectrum of adducts formed between APAP-dosed mice of any genotype. The cumulative data suggest that Cyp1a2 doses not play a significant role in APAP hepatotoxicity in these mice.