Species differences in metabolic activation and inactivation of 1-nitropyrene in the liver

To extrapolate from animal studies to humans the risk of 1-nitropyrene (1-NP), we determined the differences between human and experimental animals in oxidative activation of 1-NP to 1-NP oxides and inactivation of 1-NP oxides by epoxide hydration and glutathione conjugation in hepatic subcellular fractions from 6 species including humans. Species differences were found in both activation of 1-NP and inactivation of 1-NP oxides. 1-Nitro-4,5-dihydro-4,5-epoxypyrene-producing activity was highest in guinea pig and dog, followed by hamster, rat, human, and mouse. 1-Nitro-9,10-dihydro-9,10-epoxypyrene-producing activity was highest in hamster, followed in order by guinea pig, rat, dog, mouse, and human. The ratio of 1-nitro-4,5-dihydro-4,5-epoxypyrene to 1-nitro-9,10-dihydro-9,10-epoxypyrene also varied with the animal species. Hydration of 1-nitro-4,5-dihydro-4,5-epoxypyrene was highest in human, followed by dog, guinea pig, hamster, rat, and mouse. 1-nitro-9,10-dihydro-9,10-epoxypyrene was a poor substrate for epoxide hydrolase in all species. Glutathione conjugation of 1-NP oxides in rodents was higher than that in human and dog. In humans, hepatic microsomes produced the lowest level of 1-NP oxides but hydrolyzed them most efficiently, and glutathione conjugation activity of the cytosol was as low as in dogs, and there was a wide degree of interindividual variations in these activities. No single species studied was a good model for humans, and the balance of activation/inactivation tends toward detoxification in these adult animals.     

Effect of modifiers of the cytochrome P-450-dependent enzyme system of the liver on the metabolic pathways of diethylnitrosamine activation and inactivation

Pretreatment of Wistar male rats with antioxidants prevented the toxic effect of diethylnitrosamine (DENA) at LD50. Six-fold acceleration of DENA excretion and significant increase of maximum plasma concentration of a DENA metabolite nitrite were, also observed after antioxidants treatment. Liver microsomal metabolism of DNA was altered by pretreatment with another antioxidant--butylhydroxytoluene, which stimulated selectively denitrosation and inhibited dealkylation of DENA in the microsomal cytochrome P-450-dependent enzyme system. Moreover, butylhydroxytoluene treatment diminished he ability of microsomes to activate DENA to mutagenic intermediates identified in Ames' test. It was suggested that the protective effect of antioxidants against DENA toxicity may be due to the acceleration of its metabolic inactivation and the inhibition of its activation in liver cytochrome P-450-dependent systems.     

Multiple metabolic pathways for the mutagenic activation of 3-nitrobenzo[a]pyrene

Rat liver microsomal metabolism of the potent mutagen 3-nitrobenzo[a]pyrene(3-nitro-BaP) under aerobic conditions yielded 3-nitro-BaP trans-7,8-dihydrodioland 3-nitro-BaP trans-9,10-dihydrodiol, and under anaerobicconditions produced 3-amino-BaP. All of these metabolites werehighly mutagenic in the Salmonella typhimurium reversion assayboth with and without exogenous metabolic activation (S9). Noneof the in vitro metabolic pathways led to detoxification of3-nitro-BaP.     

Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells

The heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine (PhIP) are pyrolysis products formed when meat is cooked and are rodent mammary carcinogens. They are thought to be metabolically activated by N-hydroxylation, catalysed by cytochrome P450 (CYP), followed by O-acetylation catalysed by N- acetyltransferases. Primary cultures of human mammary epithelial cells (HMECs) prepared from up to 26 individuals for each compound, were treated with IQ, MeIQ, or PhIP (500 microM) or with N-hydroxy-2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) or N-hydroxy-2-amino- 3-methylimidazo[4,5-f]quinoline (N-OH-IQ) (20 microM) and the levels of adduct formation in their DNA analysed by 32P-post-labelling. In order to investigate whether pharmacogenetic polymorphisms influence DNA adduct formation, the NAT2 genotype of each individual was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that distinguishes between the wild-type and four variant alleles. Presence of two variant alleles designates a slow NAT2 acetylator, whereas individuals with one or two wild-type alleles are designated fast NAT2 acetylators. Interindividual variations in total DNA adduct levels ranged for IQ from 0.64-63.1 DNA adducts per 10(8) nucleotides (mean 7.80), for MeIQ from 1.99-17.8 (mean 6.63), for PhIP from 0.13-4.0 (mean 0.96), for N-OH-PhIP from 6.32-497 (mean 176) and for N-OH-IQ from 0.92-30.6 (mean 9.24). The higher adduct levels observed in cells treated with the N-OH metabolites suggests that N- hydroxylation is the rate-limiting step in HMECs and this may be due to low CYP levels. In contrast, the Phase II reaction catalysed by N- acetyltransferases is probably the major step in the metabolic activation of heterocyclic amines that occurs in the breast. Higher mean levels of heterocyclic amine-DNA adduct formation were detected in the cells of NAT2 fast acetylators compared with slow acetylators, with mean adduct levels per 10(8) nucleotides following IQ treatment, of 12.74 and 3.57 respectively, following PhIP treatment, of 1.20 and 0.74, respectively, following MeIQ treatment, of 7.90 and 5.08, respectively and following N-OH-PhIP-treatment, of 243.1 and 130.0, respectively. However, due to the large variations in adduct levels, these differences in mean values were not statistically significant with the limited number of individuals studied. This appears to be the first pilot study to demonstrate interindividual variations in the metabolic activation of heterocyclic amines and their metabolic intermediates in primary cultures of HMECs in vitro.      

Enzymatic mechanisms in the metabolic activation of N-nitrosodialkylamines

The metabolism of several N-nitrosodialkylamines was studied using rat liver microsomes and purified cytochrome P450 isozymes in a reconstituted monooxygenase system. With purified acetone/ethanol-inducible cytochrome P450 (P450ac), high N-nitrosodimethylamine (NDMA) demethylase activity was observed. Cytochrome b5 was also involved in NDMA metabolism by decreasing the Km of NDMA demethylase. A close relationship between the demethylation and denitrosation of this substrate was observed. P450ac was also active in the metabolism of N-nitrosoethylmethylamine (NEMA), but was less active than phenobarbital-inducible cytochrome P450 (P450b) in the metabolism of N-nitrosobutylmethylamine (NBMA), especially in catalysing the debutylation reaction. Similar substrate specificity was demonstrated with liver microsomes from rats treated with other inducers. With different P450 isozymes and microsomes, a close relationship between metabolism and activation of nitrosamines to mutagens to V79 cells was demonstrated. DNA alkylation by NDMA in vitro was correlated with the rate of metabolism of these compounds, whereas DNA alkylation in vivo was more complex and was dose-dependent. The work demonstrates the importance of knowledge of the substrate specificity of cytochrome P450 isozymes in understanding the mechanisms of the metabolic activation of nitrosamines.     

Individual variation and dose dependency in the progression rate of skin telangiectasia

The progression rate of late skin telangiectasia after radiotherapy has been studied prospectively in patients for various fractionation schedules and dose levels. The degree of telangiectasia was scored on an arbitrary scale ranging from no detectable to totally confluent telangiectasia. Skin telangiectasia showed a progressive development at least up to 10 years. The rate of progression was strongly dose dependent. This finding has two important implications: the dose-latency relationship is steep, and the steepness of the dose-response relationship increases with the follow-up time. The most striking finding in this study was that the individual variation in progression rate was very large for the same treatment with a documented small variation in dose. For example, the latency for telangiectasia score 2 ranged between 17 and 90 months after 35 fractions of 1.8 Gy. The reasons for the large individual variation in progression rate are unclear and will be investigated further.     

Generation of free radicals and induction of DNA adducts by activation of heterocyclic aromatic amines via different metabolic pathways in vitro

Food-derived heterocyclic aromatic amines (HCAs) have proved to be carcinogenic in both rodents and nonhuman primates. Two different metabolic pathways are suggested for the metabolic activation of HCA. The hepatic pathway proceeds via a two-step process involving N-hydroxylation by cytochrome P4501A2 and subsequent O-acetylation by N-acetyltransferase-2. An alternative pathway may be of particular interest in extrahepatic tissues and proceeds via one-electron oxidation catalyzed by prostaglandin H synthase (PHS), rendering free-radical metabolites. In this study, we investigated the metabolic activation of two HCAs, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), by two different enzyme systems in vitro, generating different primary and secondary reactive metabolites. Rat liver S9 mix and PHS were used as the activating system and represent the hepatic and extrahepatic pathways, respectively. Electron-spin resonance spectroscopy showed that both IQ and PhIP exerted inhibiting effects on PHS-mediated formation of hydroxyl radicals during the conversion of arachidonic acid to prostaglandins. Evidence for the formation of HCA free radicals was presented in an indirect way by the formation of glutathione-derived thiyl radicals, with purified PHS as the activating system. Activation by S9 mix did not result in the formation of detectable radical metabolites, showing that the two metabolic routes primarily led to the formation of different metabolites. In all electron-spin resonance experiments, IQ appeared to be more effective than PhIP. In contrasts, DNA adduct analysis by means of (32)P-postlabeling showed similar adduct patterns for S9 and PHS in single-stranded and double-stranded salmon testes DNA after incubation with PhIP, indicating the ultimate formation of a common reactive intermediate. For IQ, activation by PHS led to an additional adduct spot that was not present after S9 activation. Furthermore, activation of IQ resulted in higher adduct levels compared with PhIP for both activation pathways. Overall, adduct levels were higher in single-stranded DNA than double-stranded DNA. Our results showed that the hepatic and extrahepatic pathways resulted in different primary metabolites, while the ultimate formation of a similar reactive intermediate for PhIP, possibly an arylnitrenium ion, suggested that both pathways could play an important role in the onset of carcinogenesis.     

Role of N-methylolpentamethylmelamine in the metabolic activation of hexamethylmelamine

Hexamethylmelamine (HMM) is metabolized by rat hepatic microsomal preparations to reactive species which covalently bind to microsomal protein and to calf thymus DNA added to microsomal incubation mixtures. Covalent binding to macromolecules is dependent on the presence of molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate and is catalyzed by cytochrome P-450 monooxygenases. Reduced nicotinamide adenine dinucleotide-dependent covalent binding of [methyl-14C]HMM to microsomal protein is greater than that of [ring-14C]HMM. Reduced nicotinamide adenine dinucleotide phosphate-dependent covalent binding of [ring-14C]HMM and [methyl-14C]HMM to calf thymus DNA added to microsomal incubation mixtures are approximately equal. The [ring-14C]-labeled carbinolamine intermediate in HMM demethylation, N-methylolpentamethylmelamine, covalently binds to microsomal protein and, to a much greater extent, to calf thymus DNA.     

N-demethylation accompanies alpha-hydroxylation in the metabolic activation of tamoxifen in rat liver cells.

Previous work has shown that a major route of activation of tamoxifen to DNA-binding products in rat liver cells is via alpha-hydroxylation leading to modification of the N(2)-position of guanine in DNA and to a lesser extent the N(6)-position of adenine. Improved resolution by HPLC has now identified two major adducts in rat liver DNA, one of them the aforementioned tamoxifen-N(2)-guanine adduct and the other the equivalent adduct in which the tamoxifen moiety has lost a methyl group. Treatment of rats or rat hepatocytes with N-desmethyltamoxifen gave rise to the second adduct, whereas treatment with tamoxifen or alpha-hydroxytamoxifen gave rise to both. Furthermore, N,N-didesmethyltamoxifen was found to be responsible for an additional minor DNA adduct formed by tamoxifen, alpha-hydroxytamoxifen and N-desmethyltamoxifen. The involvement of metabolism at the alpha position was confirmed in experiments in which [alpha-D(2)-ethyl]tamoxifen, but not [beta-D(3)-ethyl]tamoxifen, produced reduced levels of DNA adducts. Tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide also gave rise to DNA adducts in rat liver cells, but the adduct patterns were very similar to those formed by tamoxifen and alpha-hydroxytamoxifen, indicating that the N-oxygen is lost prior to DNA binding. These and earlier results demonstrate that in rat liver cells in vivo and in vitro, Phase I metabolic activation of tamoxifen involves both alpha-hydroxylation and N-demethylation, which is followed by Phase II activation at the alpha-position to form a highly reactive sulphate. Detection of tamoxifen-related DNA adducts by (32)P-postlabelling is achieved with >90% labelling efficiency.     

The study of metabolic pathways in tumors based on the transcriptome

DNA microarray technology revolutionized gene-expression analysis in molecular biology to observe patterns of gene expression in genomic scale. We review the biological aspects of genome-wide gene-expression activity in tumors specially focusing on the analysis of enzyme coding genes. First, the methods for analyzing gene-expression data for the study of metabolome in silico are discussed showing SV40T antigen expressing liver tumor data as an example. Next, an application for tumor metabolome analysis utilizing a reference set of gene-expression profiles is shown.     

Immortalization of oral keratinocytes by functional inactivation of the p53 and pRb pathways

A subgroup of head and neck squamous cell carcinomas (HNSCCs) contains high‐risk human papillomavirus‐type 16 (HPV16). The viral E6 and E7 oncoproteins inactivate the p53 and pRb proteins, respectively. We examined the causative effect of HPV16 E6 and E7 expression on the immortalization of normal oral keratinocytes (OKCs) and compared the resulting phenotype with alternative ways of p53‐ and pRb‐pathway abrogation frequently found in HNSCCs without HPV. Primary OKCs were conditionally immortalized with temperature‐sensitive SV40 large T‐antigen and human telomerase, allowing these cells to return to their senescent primary state after temperature shift. HPV16 E6 and E7 were introduced to overcome senescence, determined with population doubling (PD) as read‐out. For comparison, we downregulated p53 and p16 by short hairpin RNA genes and expressed mutant p53R(175)H and cyclinD1. Expression of HPV16 E6 caused an extended life span similar to expression of mutant p53R(175)H or p53 knockdown. Expression of mutant p53R(175)H seemed to cause additional activation of the hypoxia and WNT signaling pathways. HPV16 E7 expression had no direct effect on lifespan, similar to p16 knockdown or cyclinD1 expression. In combination with HPV16 E6 or other functional inactivations of p53, abrogation of the pRb‐pathway by either HPV16 E7 or other manipulations caused an immortal phenotype. Our data show the causative role of HPV16 E6/E7 in early squamous carcinogenesis. Activity of each gene could be mimicked by other genetic events frequently found in HNSCC without HPV. This data provides the experimental proof of causal association of HPV in HNSCC carcinogenesis and further support the crucial role of the p53‐ and pRb‐pathways.     

The metabolism of cyclophosphamide. Dose dependency and the effect of long-term treatment with cyclophosphamide.

Studies on the metabolism of cyclophosphamide-14C were performed in 10 subjects at different single dose levels within the range of 0.02-10 mg/kg body-weight and in five subjects before and following an average 22 days treatment with cyclophosphamide in daily doses of 2 mg/kg. The parameters of cyclophosphamide metabolism--serum half-life of unchanged cyclophosphamide, serum concentration of metabolites, and rates of excretion of cyclophosphamide and metabolites in the urine--were all independent of dose. No change of the metabolism was demonstrated after treatment with cyclophosphamide for 22 days.     

In vitro activation of cyclophosphamide for an in vitro chemosensitivity assay

The problem of activation of cyclophosphamide (CY) for an in vitro chemosensitivity assay was studied using the B16 melanoma. The efficacy of the S9 hepatic microsomal fraction in vitro was compared with activation by passage of drug in vivo. The effect of CY was assayed by inhibition of tritiated thymidine (³HdThd) uptake by B16 tumor cells in vitro, and by its effect in single dose in vivo on the life span of syngeneic C57BL/6 mice injected with B16 tumor cells. In vivo activation of CY that was achieved by injecting 20 mg of CY intraperitoneally (i.p.) into mice and obtaining plasma 20 minutes later was more rapid and more reproducible, while in vitro activation provided a more potent preparation and a better quantitative correlation with the in vivo effect of CY. The activation activity of different preparations of S9 microsomal fractions was found to be directly related to the activity of aminopyrine demethylase, a mixed function oxidase enzyme, in the S9 fraction, rather than to total protein concentration. The S9-activated drug required 2 1/2 to 3 hours to achieve maximum inhibition of subsequent tumor cell tritiated thymidine (³HdThd) incorporation compared with 20 minutes to achieve maximum inhibition by in vivo activated drug. We conclude that rapid qualitative screening for effectiveness of CY may best be done with in vivo activated drug, whereas quantitative prediction of effective concentration appears to be best achieved with drug activated in vitro by the hepatic S9 fraction.     

Genetic susceptibility according to three metabolic pathways in cancers of the lung and bladder and in myeloid leukemias in nonsmokers.

BACKGROUND: We chose a set of candidate single nucleotide polymorphisms (SNPs) to investigate gene-environment interactions in three types of cancer that have been related to air pollution (lung, bladder and myeloid leukemia). PATIENTS AND METHODS: The study has been conducted as a nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort (409 cancer cases and 757 matched controls). We included never and ex-smokers. SNPs were in genes involved in oxidative stress, phase I metabolizing genes, phase II metabolizing genes and methylenetetrahydrofolate reductase (MTHFR). RESULTS: The most notable findings are: GSTM1 deletion and bladder cancer risk [odds ratio (OR) = 1.60; 95% confidence interval 1.00-2.56]; CYP1A1 and leukemia (2.22, 1.33-3.70; heterozygotes); CYP1B1 and leukemia (0.47, 0.27-0.84; homozygotes); MnSOD and leukemia (1.91, 1.08-3.38; homozygotes) and NQO1 and lung cancer (8.03, 1.73-37.3; homozygotes). Other statistically significant associations were found in subgroups defined by smoking habits (never or ex-smokers), environmental tobacco smoke or gender, with no obvious pattern. When gene variants were organized according to the three main pathways, the emerging picture was of a strong involvement of combined phase I enzymes in leukemia, with an OR of 5 (1.63-15.4) for those having three or more variant alleles. The association was considerably stronger for leukemias arising before the age of 55.     

Of spiders and crabs: the emergence of lysophospholipids and their metabolic pathways as targets for therapy in cancer.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), two small lysophospholipids, are potent inducers of many of the hallmarks of cancer including cell proliferation, survival, migration, invasion, and neovascularization in in vitro and in vivo tumor models. Furthermore, the enzymes metabolizing LPA and S1P and their receptors are aberrant in multiple cancer lineages and exhibit transforming activity altering patterns and targets for metastasis. Several recent studies show the remarkable activity of new chemical genomics and/or potential novel drugs in preclinical models. Combined with the physiologic and pathophysiologic activities of LPA and S1P, these studies suggest the implementation of preclinical and clinical evaluation of LPA and S1P as therapeutic targets.     

Cytotoxicity of cyclophosphamide in the rat incisor.

Three of the 4 groups of 3 Wistar rats each were given 40 mg, 80 mg and 120 mg cyclophosphamide/kg respectively by single intraperitoneal injections. The fourth group was given 2 ml of normal saline as control. One animal from each group was killed after 1, 4 and 8 days. The incisor teeth of all experimental animals showed evidence of cytotoxic injury, which appeared to be more severe with increasing dosage, to the undifferentiated mesenchymal cells in the proliferating zone of the pulp close to the basal odontogenic epithelium, cessation of root growth and relative acellularity of the basal area of the pulp. Evidence of cytotoxicity to the odontogenic epithelium was seen only in the groups given 80 mg/kg and 120 mg/kg. Resolution of the cytotoxic injury and re-establishment of normal basal odontogenesis were seen in the 40 mg dose group by the eighth day but appeared to be slower with increasing dosage. It would seem that of the rapidly proliferating epithelial and mesenchymal odontogenic cells in the basal area of the rat incisor those in the mesenchyme may be most susceptible to the cytotoxicity of cyclophosphamide. The odontogenic epithelium may be resistant to the cytotoxicity of 40 mg cyclophosphamide/kg. The results may be of significance in the investigation of the mechanism of cytotoxicity of this cancer chemotherapeutic agent.