Lung Microsomes Research Articles

Identification of cytochrome P450 enzymes critical for lung tumorigenesis by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): insights from a novel Cyp2abfgs-null mouse.

Cytochrome P450 (P450) enzymes encoded by the mouse Cyp2abfgs gene cluster are preferentially expressed in the respiratory tract. Previous studies have demonstrated that pulmonary P450-mediated bioactivation is necessary for lung tumorigenesis induced by the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and that CYP2A5 mediates a noteworthy fraction, but not all, of NNK bioactivation in the lung. The aim of this study was to determine whether other P450s encoded by the Cyp2abfgs gene cluster also play significant roles in NNK lung tumorigenesis. A novel Cyp2abfgs-null mouse was generated, in which all Cyp2a, 2b, 2g, 2f and 2s genes are deleted. The Cyp2abfgs-null mouse was viable, fertile and without discernible physiological abnormalities or compensatory increases in the expression of other P450s. NNK bioactivation in vitro and NNK-induced DNA adduction and lung tumorigenesis in vivo were determined for wild-type (WT) and Cyp2abfgs-null mice; the results were compared with previous findings from Cyp2a5-null mice. The Cyp2abfgs-null mice exhibited significantly lower rates of NNK bioactivation in lung and liver microsomes, compared with either WT or Cyp2a5-null mice. The levels of lung O(6)-methyl guanine DNA adduct were also substantially reduced in Cyp2abfgs-null mice, compared with either WT or Cyp2a5-null mice. Moreover, the Cyp2abfgs-null mice were largely resistant to NNK-induced lung tumorigenesis at both low (50mg/kg) and high (200mg/kg) NNK doses, in contrast to the WT or Cyp2a5-null mice. These results indicate for the first time that, collectively, the CYP2A, 2B, 2F, 2G, and 2S enzymes are indispensable for NNK-induced lung tumorigenesis.

Metabolism of styrene to styrene oxide and vinylphenols in cytochrome P450 2F2- and P450 2E1-knockout mouse liver and lung microsomes.

Pulmonary toxicity of styrene is initiated by cytochromes P450-dependent metabolic activation. P450 2E1 and P450 2F2 are considered to be two main cytochrome P450 enzymes responsible for styrene metabolism in mice. The objective of the current study was to determine the correlation between the formation of styrene metabolites (i.e., styrene oxide and 4-vinylphenol) and pulmonary toxicity of styrene, using Cyp2e1- and Cyp2f2-null mouse models. A dramatic decrease in the formation of styrene glycol and 4-vinylphenol was found in Cyp2f2-null mouse lung microsomes relative to that in the wild-type mouse lung microsomes; however, no significant difference in the production of the styrene metabolites was observed between lung microsomes obtained from Cyp2e1-null and the wild-type mice. The knockout and wild-type mice were treated with styrene (6.0 mmol/kg, ip), and cell counts and LDH activity in bronchoalveolar lavage fluids were monitored to evaluate the pulmonary toxicity induced by styrene. Cyp2e1-null mice displayed a susceptibility to lung toxicity of styrene similar to that of the wild-type animals; however, Cyp2f2-null mice were resistant to styrene-induced pulmonary toxicity. In conclusion, both P450 2E1 and P450 2F2 are responsible for the metabolic activation of styrene. The latter enzyme plays an important role in styrene-induced pulmonary toxicity. Both styrene oxide and 4-vinylphenol are suggested to participate in the development of lung injury induced by styrene.

Role of CYP2A13 in the bioactivation and lung tumorigenicity of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: in vivo studies using a CYP2A13-humanized mouse model.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is abundant in tobacco smoke, is a potent lung procarcinogen. The present study was aimed to prove that transgenic expression of human cytochrome P450 2A13 (CYP2A13), known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, will enhance NNK bioactivation and NNK-induced tumorigenesis in the mouse lung. Kinetic parameters of NNK bioactivation in vitro and incidence of NNK-induced lung tumors in vivo were determined for wild-type, Cyp2a5-null and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. As expected, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in Michaelis constant (K m) values for the formation of 4-oxo-4-(3-pyridyl)-butanal, representing the reactive intermediate that can lead to the formation of O(6)-methylguanine (O(6)-mG) DNA adducts; however, the gain of CYP2A13 at a fraction of the level of mouse lung CYP2A5 led to recovery of the activity in the lung, but not in the liver. The levels of O(6)-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of CYP2A13-humanized mice than in Cyp2a5-null mice. Moreover, incidences of lung tumorigenesis were significantly greater in CYP2A13-humanized mice than in Cyp2a5-null mice, and the magnitude of the differences in incidence was greater at low (30mg/kg) than at high (200mg/kg) NNK doses. These results indicate that CYP2A13 is a low K m enzyme in catalyzing NNK bioactivation in vivo and support the notion that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans.

Benzylmorpholine Analogs as Selective Inhibitors of Lung Cytochrome P450 2A13 for the Chemoprevention of Lung Cancer in Tobacco Users

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer. Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is a major challenge in compound design. A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6. Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure.

Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues

1. Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure–metabolic activity relationship was examined.2. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains.3. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben.4. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity.5. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes.6. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens.7. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.

Comparative in vitro metabolism of phospho-tyrosol-indomethacin by mice, rats and humans

Phospho-tyrosol-indomethacin (PTI; MPI 621), a novel anti-cancer agent, is more potent and safer than conventional indomethacin. Here, we show that PTI was extensively metabolized in vitro and in vivo. PTI was rapidly hydrolyzed by carboxylesterases to generate indomethacin as its major metabolite in the liver microsomes and rats. PTI additionally undergoes cytochromes P450 (CYP)-mediated hydroxylation at its tyrosol moiety and O-demethylation at its indomethacin moiety. Of the five major human CYPs, CYP3A4 and CYP2D6 catalyze the hydroxylation and O-demethylation reactions of PTI, respectively; whereas CYP1A2, 2C9 and 2C19 are inactive towards PTI. In contrast to PTI, indomethacin is primarily O-demethylated by CYP2C9, which prefers acidic substrates. The hydrolyzed and O-demethylated metabolites of PTI are further glucuronidated and sulfated, facilitating drug elimination and detoxification. We observed substantial inter-species differences in the metabolic rates of PTI. Among the liver microsomes from various species, PTI was the most rapidly hydrolyzed, hydroxylated and O-demethylated in mouse, human and rat liver microsomes, respectively. These results reflect the differential expression patterns of carboxylesterase and CYP isoforms among these species. Of the human microsomes from various tissues, PTI underwent more rapid carboxylesterase- and CYP-catalyzed reactions in liver and intestine microsomes than in kidney and lung microsomes. Together, our results establish the metabolic pathways of PTI, reveal significant inter-species differences in its metabolism, and provide insights into the underlying biochemical mechanisms.

마이크로웨이브를 조사한 흰쥐 폐조직의 Phospholipae A2활성과 Arachidonic Acid Cascade계에 미치는 녹차 Catechin의 영향

본 연구에서는 흰쥐에 마이크로웨이브를 조사한 후 폐조직을 대상으로 폐조직 기능장애를 일으키는데 주된 역할을 하는 혈전생성능을 arachidonic acid(AA) cascade계를 통해 관찰하여 마이크로웨이브에 의한 폐혈관 기능장애와 그에 대한 녹차 catechin의 항혈전 효과를 규명하고자 하였다. 실험군은 마이크로웨이브를 조사하지 않은 정상군과 마이크로웨이브를 조사한 군으로 나누고 마이크로웨이브 조사 군은 다시 식이 중 catechin 공급수준에 따라 catechin을 넣지 않은 군(MW group), catechin을 0.25% 급여한 군(MW-0.25C group), catechin을 0.5% 급여한 군(MW-0.5C group)으로 나누었다. 식이와 음료는 자유 섭식시키면서 2주간 사육한 후 2.45 GHz 대역의 주파수를 15분간 1회 조사하였으며 마이크로웨이브 조사 후 6일째 동물을 희생시켜 본 실험에 사용하였다. 실험군의 실험동물 수는 각각 10마리로 실험하였다. PLA2 활성은 마이크로웨이브 조사로 30% 증가하였으며 MW-0.25C군은 15% 증가였으나 MW-0.5C군은 정상군 수준이었다. 인지질분자종의 변화를 관찰한 결과 lyso PE가 MW군에서 47% 증가되었으나 MW-0.25C군 및 MW-0.5C군에서는 각각 18%, 20% 증가되었다. TXA₂ 생성은 MW군에서 50%의 현저한 증가를 보였으나 catechin 공급군인 MW-0.25C군 및 MW-0.5C군은 정상군 수준이었다. PGI₂ 생성은 MW군에서 31%의 유의적인 감소를 보였으나 catechin 공급군인 MW-0.25C군 및 MW-0.5군은 정상군 수준이었다. 따라서 혈전생성지표인 PGI2/TXA2 ratio는 정상군에 비해 MW군에서 43% 유의적으로 감소되었으나 MW-0.25C군 및 MW-0.5C군은 정상군 수준이었다. 지질과산화물의 함량은 MW군에서 34% 유의적으로 증가하였으며 catechin 공급군은 정상군 수준이었다. 결론적으로 마이크로웨이브에 피폭된 흰쥐 폐조직에서는 AA cascade계의 율속 효소인 PLA₂ 활성의 증가와 혈전생성지표로 인식하는 PGI₂/TXA₂ ratio의 불균형이 초래되었으나 catechin은 TBARS 농도를 낮추면서 PLA₂ 활성을 저해시키고 AA cascade계를 개선시킴으로써 항혈전 작용을 나타내었다.

Kinetic modeling of β-chloroprene metabolism: Probabilistic in vitro–in vivo extrapolation of metabolism in the lung, liver and kidneys of mice, rats and humans

β-Chloroprene (chloroprene) is carcinogenic in inhalation bioassays with B6C3F1 mice and Fischer rats, but the potential effects in humans have not been adequately characterized. In order to provide a better basis for evaluating chloroprene exposures and potential effects in humans, we have explored species and tissue differences in chloroprene metabolism. This study implemented an in vitro–in vivo extrapolation (IVIVE) approach to parameterize a physiologically based pharmacokinetic (PBPK) model for chloroprene and evaluate the influence of species and gender differences in metabolism on target tissue dosimetry. Chloroprene metabolism was determined in vitro using liver, lung and kidney microsomes from male or female mice, rats, and humans. A two compartment PK model was used to estimate metabolism parameters for chloroprene in an in vitro closed vial system, which were then extrapolated to the whole body PBPK model. Two different strategies were used to estimate parameters for the oxidative metabolism of chloroprene: a deterministic point-estimation using the Nelder-Mead nonlinear optimization algorithm and probabilistic Bayesian analysis using the Markov Chain Monte Carlo technique. Target tissue dosimetry (average amount of chloroprene metabolized in lung per day) was simulated with the PBPK model using the in vitro-based metabolism parameters. The model-predicted target tissue dosimetry, as a surrogate for a risk estimate, was similar between the two approaches; however, the latter approach provided a measure of uncertainty in the metabolism parameters and the opportunity to evaluate the impact of that uncertainty on predicted risk estimates.

Abstract 5465: Role of CYP2A13 in NNK bioactivation and lung tumorigenesis: In vivo studies using a CYP2A13-humanized mouse model

Abstract The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung procarcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis depends on target tissue bioactivation by pulmonary P450 enzymes (Weng et al., 2007, Cancer Res 67:7825-7832). However, the specific P450 enzyme(s) responsible for NNK bioactivation in the lung have not been identified. The aim of the present study was to test the hypotheses that 1) mouse CYP2A5 plays an essential role in NNK bioactivation and tumorigenesis in mouse lung, and 2) transgenic expression of human CYP2A13, known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, can increase the rates of NNK bioactivation and incidence of tumorigenesis in the mouse lung. Kinetic parameters of microsomal NNK bioactivation in vitro, tissue levels of O6-methylguanine (O6-mG) DNA adducts formed in vivo, and incidence of NNK-induced lung tumors were determined for wild-type, Cyp2a5-null, and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. The results indicated that, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in the Km value for the formation of 4-oxo-4-(3-pyridyl)-butanal (OPB), which represents the reactive intermediate that can lead to the formation of O6-mG DNA adducts; but, the gain of CYP2A13 led to recovery of the activity in the lung, but not in the liver. The levels of O6-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of the wild-type mice and CYP2A13-humanized mice, than in the Cyp2a5-null mice. Furthermore, lung tumor bioassays, performed for the three mouse strains on A/J background, and using NNK at various doses (30 -200 mg/kg; single i.p. dose), revealed that lung tumor multiplicity determined at 4 months post-treatment was significantly greater in wild-type and CYP2A13-humanized mice than in Cyp2a5-null mice, for all NNK doses tested. The difference in lung tumor multiplicity between CYP2A13-humanized mice and Cyp2a5-null mice was greater at low (30 mg/kg) NNK dose (∼70%) than at high (200 mg/kg) NNK dose (∼30%). In control experiments, rates of systemic clearance of NNK and its major circulating metabolite, NNAL, were confirmed to be not different among the three mouse strains. These results indicated that CYP2A13 is a low-Km enzyme in catalyzing NNK bioactivation in vivo, and they provide strong support for the idea that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans. Moreover, the CYP2A13-humanized mouse model will be valuable for testing new, CYP2A13-targeted chemopreventive agents for lung carcinogenesis. (Supported in part by NIH grant CA092596) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5465. doi:1538-7445.AM2012-5465

Relationship of Metabolism and Cell Proliferation to the Mode of Action of Fluensulfone-Induced Mouse Lung Tumors: Analysis of Their Human Relevance Using the IPCS Framework

Species-specific lung tumors in the mouse are induced by a number of chemicals. The underlying cause appears to be a high metabolic activity of mouse lung, due to relatively high abundance of Clara cells in mice compared with humans and the mouse-specific cytochrome P450 isoform 2f2 in the Clara cells. The chemicals are activated to reactive intermediates, leading to local cytotoxicity or mitogenicity resulting in increased cell proliferation and tumors. Rats have lower metabolic activity than mice (already below the threshold needed to cause lung tumors upon lifetime exposure) and activity in humans is lower than in rats. The carcinogenic risk for human lung is low for this mode of action (MOA). Fluensulfone has shown an increased incidence of lung adenomas in mice, but not in rats, at high doses. Fluensulfone is not genotoxic. MOA studies were conducted investigating key events of the postulated MOA. Fluensulfone is extensively metabolized by mouse lung microsomes, whereas no metabolic activity is seen with human lung microsomes. Cyp 2f2 is a major contributor in fluensulfone’s metabolism and Cyp 2e1 is not involved. Furthermore, administration of fluensulfone to mice led to an early increase in Clara cell proliferation. The International Programme on Chemical Safety (IPCS) MOA and human relevance framework was used to evaluate the collective data on fluensulfone. We concluded that fluensulfone leads to species-specific mouse lung tumors and that these tumors are likely not relevant to human hazard or risk.

Biotransformation of GS‐1101 (CAL‐101), a Potent and Selective Inhibitor of PI3K delta for the Treatment of Patients with Hematologic Malignancies

The objective of this study was to study the biotransformation of GS‐1101 and its metabolites in humans in vitro. Abundant mono‐oxygenated GS‐1101 (GS‐563117 ) was detected by LC/MS/MS in human clinical samples, suggesting oxidation is a major pathway for disposition. Structure elucidation of GS‐563117 revealed the purine moiety to be the principal oxidation site. In vitro approaches were employed to study the metabolism of GS‐1101 to GS‐563117. Oxidation was primarily catalyzed by CYP3A4/5 although xanthine oxidase (XO) also catalyzed the reaction. Biotransformation was prominent in liver microsomes and to a lesser extent in small intestinal, renal, and lung microsomes. In addition to direct excretion, clinical studies indicated that GS‐563117 is also eliminated via glucuronidation. In vitro studies demonstrated that the reaction is primarily catalyzed by UGT1A4. In conclusion, CYP3A4/5‐catalyzed mono‐oxygenation is a primary biotransformation reaction of GS‐1101 although XO also participates in catalysis of the reaction in vitro. In vitro data also indicated that GS‐563117 is further metabolized via UGT1A4‐catalyzed glucuronidation.

Maternal intake of quercetin during gestation alters ex vivo benzo[a]pyrene metabolism and DNA adduct formation in adult offspring

Variation in xenobiotic metabolism cannot entirely be explained by genetic diversity in metabolic enzymes. We suggest that maternal diet during gestation can contribute to variation in metabolism by creating an in utero environment that shapes the offspring's defence against chemical carcinogens. Therefore, pregnant mice were supplemented with the natural aryl hydrocarbon receptor (AhR) agonist quercetin (1 mmol quercetin/kg feed) until delivery. Next, it was investigated whether the adult offspring at the age of 12 weeks had altered biotransformation of the environmental pollutant benzo[a]pyrene (B[a]P). In utero quercetin exposure resulted in significantly enhanced gene expression of Cyp1a1, Cyp1b1, Nqo1 and Ugt1a6 in liver of foetuses at Day 14.5 of gestation. Despite cessation of supplementation after delivery, altered gene expression persisted into adulthood, but in a tissue- and gender-dependent manner. Expression of Phase I enzymes (Cyp1a1 and Cyp1b1) was up-regulated in the liver of adult female mice in utero exposed to quercetin, whereas expression of Phase II enzymes (Gstp1, Nqo1 and Ugt1a6) was predominantly enhanced in the lung tissue of female mice. Epigenetic mechanisms may contribute to this adapted gene expression, as the repetitive elements (SINEB1) were hypomethylated in liver of female mice prenatally exposed to quercetin. Studies on ex vivo metabolism of B[a]P by lung and liver microsomes showed that the amount of B[a]P-9,10-dehydrodiol, B[a]P-7,8-dihydrodiol and 3-hydroxy-B[a]P did not change, but the amount of unmetabolised B[a]P was significantly lower after incubation with lung microsomes from offspring that received quercetin during gestation. Moreover, ex vivo B[a]P-induced DNA adduct formation was significantly lower for liver microsomes of offspring that were exposed to quercetin during gestation. These results suggest that prenatal diet leads to persistent alterations in Phase I and II enzymes of adult mice and may affect cancer risk.

Role of CYP2A5 in the Bioactivation of the Lung Carcinogen 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone in Mice

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis in mice depends on target-tissue bioactivation by pulmonary cytochrome P450 (P450) enzymes. The present study was designed to test the hypothesis that mouse CYP2A5 plays an essential role in NNK bioactivation in mouse lung. The role of CYP2A5 in NNK bioactivation was studied both in vitro and in vivo, by comparing the kinetic parameters of microsomal NNK metabolism and tissue levels of O(6)-methylguanine (O(6)-mG) (the DNA adduct highly correlated with lung tumorigenesis) between wild-type (WT) and Cyp2a5-null mice. In both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in the apparent K(m) values for the formation of 4-oxo-4-(3-pyridyl)butanone, which represents the reactive intermediate that produces O(6)-mG in vivo. The loss of CYP2A5 did not change circulating levels of NNK or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in mice treated intraperitoneally with NNK at either 20 or 100 mg/kg. However, the levels of lung O(6)-mG were significantly lower in Cyp2a5-null than in WT mice; the extent of the reduction was greater at the 20 mg/kg dose (∼40%) than at the 100 mg/kg dose (∼20%). These results indicate that CYP2A5 is the low-K(m) enzyme for NNK bioactivation in mouse lung. It is noteworthy that the remaining NNK bioactivation activities in the Cyp2a5-null mice could be inhibited by 8-methoxypsoralen, a P450 inhibitor used previously to demonstrate the role of CYP2A5 in NNK-induced lung tumorigenesis. Thus, P450 enzymes other than CYP2A5 probably also contribute to NNK-induced lung tumorigenesis in mice.

Enhanced activity of punicalagin delivered via polymeric implants against benzo[a]pyrene-induced DNA adducts

We investigated the effect of punicalagin (PC) on benzo[a]pyrene (BP)-induced DNA adducts in vitro and in vivo. Incubation of BP (1μM) with rat liver microsomes, appropriate co-factors and DNA in the presence of vehicle or punicalagin (1–40μM) showed dose-dependent inhibition of the resultant DNA adducts, with essentially complete (97%) inhibition at 40μM. However, PC failed to inhibit anti-BPDE-induced DNA adducts when tested in an in vitro non-microsomal system, suggesting that the inhibition of the microsomal BP–DNA adducts occurred due to inhibition of P450 1A1 by PC. To determine its efficacy in vivo, female S/D rats were administered punicalagin via the diet (1500ppm; ∼19mg/day/animal) or subcutaneous polymeric implants (two 2-cm, 200mg with 20% drug load; 40mg PC/implant) and then treated with continuous low-dose of BP by a subcutaneous polymeric implant (2cm, 200mg with 10% load; 20mg BP/implant) and euthanized after 10 days. Analysis of the lung DNA by 32P-postlabeling showed significant (60%; p=0.029) inhibition of DNA adducts by PC administered via the implants; the dietary route showed modest (34%) but statistically insignificant inhibition. Furthermore, total PC administered by implants was approximately 38-fold lower compared with the dietary route. Analysis of the lung microsomes showed significant inhibition of cytochrome P450 1A1 activity and induction of glutathione. Release of PC from the implants was found to be biphasic starting with a burst release, followed by a gradual decline. Ultra performance liquid chromatography analysis showed no detectable PC in the plasma but its hydrolyzed product, ellagic acid was readily detected. The plasma concentration of ellagic acid was over two orders of magnitude higher (589±78ng/mL) in the implant group compared with diet (4.36±0.83ng/mL). Together, our data show that delivery of PC by implants can reduce its effective dose substantially, and that the inhibition of DNA adducts in vivo occurred presumably due to the conversion of PC to ellagic acid.

Evaluation of bisphenol A glucuronidation according to UGT1A1*28 polymorphism by a new LC–MS/MS assay

The endocrine disruptor bisphenol A (BPA) is a frequently used chemical in the manufacture of consumer products. In humans, BPA is extensively metabolized to BPA glucuronide (BPAG) by different UDP-glucuronosyltransferase (UGT) isoforms. The study has been performed with the intention to improve the accuracy of published physiologically based pharmacokinetic models and to improve regulatory risk assessments of BPA. In order to gain insight into intestine, kidney, liver, and lung glucuronidation of BPA, human microsomes of all tested organs were used. BPAG formation followed Michaelis–Menten kinetics in the intestine and kidney, but followed substrate inhibition kinetics in the liver. Human lung microsomes did not show glucuronidation activity towards BPA. While the liver intrinsic clearance was very high (857 mL min −1 kg body weight −1), the tissue intrinsic clearances for the kidney and intestine were less than 1% of liver intrinsic clearance. Since BPA is a UGT1A1 substrate, we postulated that the common UGT1A1*28 polymorphism influences BPA glucuronidation, and consequently, BPA detoxification. Hepatic tissue intrinsic clearances for UGT1A1*1/*1, UGT1A1*1/*28, and UGT1A1*28/*28 microsomes were 1113, 1075, and 284 mL min −1 kg body weight −1, respectively. Prior to microsomal experiments, the bioproduction of BPAG and stable isotope-labeled BPAG (BPAG d16) was performed for the purpose of the reliable and accurate quantification of BPAG. In addition, a sensitive LC–MS/MS analytical method for the simultaneous determination of BPA and BPAG based on two stable isotope-labeled internal standards was developed and validated. In conclusion, our in vitro results show that the liver is the main site of BPA glucuronidation ( K m 8.9 μM, V max 8.5 nmol min −1 mg −1) and BPA metabolism may be significantly influenced by a person's genotype ( K m 10.0–13.1 μM, V max 3.4–16.2 nmol min −1 mg −1). This discovery may be an important fact for the currently on-going worldwide BPA risk assessments and for the improvement of physiologically based pharmacokinetic models.

Pulmonary Toxicity and Metabolic Activation of Tetrandrine in CD-1 Mice

Tetrandrine, a bisbenzylisoquinoline alkaloid, has demonstrated promising pharmacologic activities. The alkaloid has a great potential for clinical use, so a careful, thorough toxicity evaluation of the alkaloid is required. In the present study, 24 h acute toxicity of tetrandrine was evaluated in CD-1 mice. Single intraperitoneal doses of tetrandrine at 150 mg (0.24 mmol)/kg were found to cause alveolar hemorrhage and over 3-fold elevation of lactate dehydrogenase activity in bronchoalveolar lavage fluids. Ethidium-based staining showed loss of membrane integrity in significant numbers of cells in the lungs of the animals treated with the same doses of tetrandrine. As much as 60% reduction in cell viability was observed after 24 h of exposure to tetrandrine at 40 μM in human lung cell lines NL-20 and WI-38. Ketoconazole, an inhibitor of P450 3A, showed a protective effect on the pulmonary injury in mice given tetrandrine. A glutathione (GSH) conjugate derived from O-demethylated tetrandrine was detected in incubations of tetrandrine with NADPH- and GSH-supplemented human liver and mouse lung microsomes. The electrophilic metabolite trapped by GSH is considered to be a quinone methide derivative. The formation of the metabolite reactive to GSH was found to require the presence of NADPH. The coincubation of ketoconazole suppressed the generation of the GSH conjugate. Tetrandrine was incubated with a selection of recombinant human cytochrome P450 enzymes, and only P450s 3A4 and 3A5 were responsible for the production of the reactive metabolite. The results implicate a possible correlation between the formation of the quinone methide metabolite of tetrandrine and the pulmonary toxicity induced by tetrandrine.

Raloxifene Glucuronidation in Human Intestine, Kidney, and Liver Microsomes and in Human Liver Microsomes Genotyped for theUGT1A1*28Polymorphism

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-β-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4'-β-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min(-1) · kg(-1), respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437-444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.

Kinetics of ethylene and ethylene oxide in subcellular fractions of lungs and livers of male B6C3F1 mice and male fischer 344 rats and of human livers.

Ethylene (ET) is metabolized in mammals to the carcinogenic ethylene oxide (EO). Although both gases are of high industrial relevance, only limited data exist on the toxicokinetics of ET in mice and of EO in humans. Metabolism of ET is related to cytochrome P450-dependent mono-oxygenase (CYP) and of EO to epoxide hydrolase (EH) and glutathione S-transferase (GST). Kinetics of ET metabolism to EO and of elimination of EO were investigated in headspace vessels containing incubations of subcellular fractions of mouse, rat, or human liver or of mouse or rat lung. CYP-associated metabolism of ET and GST-related metabolism of EO were found in microsomes and cytosol, respectively, of each species. EH-related metabolism of EO was not detectable in hepatic microsomes of rats and mice but obeyed saturation kinetics in hepatic microsomes of humans. In ET-exposed liver microsomes, metabolism of ET to EO followed Michaelis-Menten-like kinetics. Mean values of Vmax [nmol/(min·mg protein)] and of the apparent Michaelis constant (Km [mmol/l ET in microsomal suspension]) were 0.567 and 0.0093 (mouse), 0.401 and 0.031 (rat), and 0.219 and 0.013 (human). In lung microsomes, Vmax values were 0.073 (mouse) and 0.055 (rat). During ET exposure, the rate of EO production decreased rapidly. By modeling a suicide inhibition mechanism, rate constants for CYP-mediated catalysis and CYP inactivation were estimated. In liver cytosol, mean GST activities to EO expressed as Vmax/Km [μl/(min·mg protein)] were 27.90 (mouse), 5.30 (rat), and 1.14 (human). The parameters are most relevant for reducing uncertainties in the risk assessment of ET and EO.

Abstract 3236: Role of mouse and human CYP2A enzymes in the bioactivation of the tobacco-specific lung procarcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Abstract NNK is a potent lung procarcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis depends on target-tissue metabolic activation by pulmonary P450 enzymes. The aim of this study is to test the hypotheses that 1) mouse CYP2A5 plays an essential role in NNK bioactivation in the lung, and 2) transgenic expression of human CYP2A13, known to be selectively expressed in the respiratory tract, and be the most efficient enzyme for NNK metabolic activation in vitro, can increase the rates of NNK metabolic activation in the mouse lung. We determined the rates of in vitro formation of 4-oxo-4-(3-pyridyl)-butanal (OPB), representing the reactive intermediate that can lead to the further formation of O6-mehtylguanine (O6-mG) DNA adduct, in liver and lung microsomes of wild-type, Cyp2a5-null, and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. We found that, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant decreases in the rates of formation of OPB; whereas, the gain of CYP2A13 led to recovery of the microsomal activity in the lung, but not in the liver. Furthermore, we observed that the levels of O6-mG, the DNA adduct known to be most highly correlated with incidences of lung tumor formation, were significantly higher in the lungs of the CYP2A13-humanized mice, than in the Cyp2a5-null mice. In control experiments, rates of systemic clearance of NNK and its major circulating metabolite, NNAL, were confirmed to be not different among the three mouse strains. These results indicate that CYP2A13 can catalyze NNK bioactivation in vivo, and they support the idea that CYP2A13 genetic polymorphisms can influence the risks of tobacco-induced lung tumorigenesis in humans. (Supported in part by NIH grant CA092596) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2011-3236

In vitro metabolism, glutathione conjugation, and CYP isoform specificity of epoxidation of 4-vinylphenol

4-Vinylphenol (4VP) has been identified as a minor urinary metabolite of styrene in rat and human volunteers. This compound has been shown to be more hepatotoxic and pneumotoxic than both styrene and styrene oxide at lower doses in rats and mice. To explore the possible toxicity mechanism of 4VP, the current study was conducted to investigate the metabolism of 4VP, the glutathione (GSH) conjugation of the metabolites of 4VP and its cytochrome P450 (CYP) specificity in epoxidation in different microsomes in vitro.Incubations of 4VP with mouse lung microsomes afforded two major metabolites which were identified as 4-(2-oxiranyl)-phenol of 4VP (4VPO) and 4VP catechol. 4VPO was found to react with GSH to form GSH conjugate and 4VP catechol was found to further be metabolized to electrophilic species which react with GSH to form the corresponding 4VP catechol GSH conjugates. Relative formation rates for those GSH conjugates and the regioisomer formation of 4VPO-GSH conjugates with both inhibitors of CYP 2F2 and CYP 2E1 in microsomal incubation condition were also investigated.This present study provides better insight on the lung toxicity seen with 4VP, the toxic metabolite of commercial styrene.