Cytochrome P450-mediated activation of phenanthrene in genetically engineered V79 Chinese hamster cells

V79 Chinese hamster cells genetically engineered for rat cytochromes P450 1A1, 1A2, 2B1 and human cytochromes P450 1A1, 1A2, 2A6, 2E1, and 3A4 are being applied in metabolism studies on polycyclic aromatic hydrocarbons. This study presents the results on phenanthrene as the prototypic polycyclic aromatic hydrocarbon possessing a bay region. Phenanthrene is of less importance regarding cytotoxicity and carcinogenicity as compared to e.g. benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene. However, phenanthrene is more readily converted to metabolites which are exreted in higher amounts than those from any other polycyclic aromatic hydrocarbon. Therefore, its metabolites are of diagnostic value in epidemiological and occupational exposure studies. For this reason, it is worthwhile to understand the metabolism of phenanthrene in detail, e.g. allocating metabolites and cytochromes P450s. In accordance to previous observations cytochromes P450 1A1 and 1A2 were the most active forms towards phenanthrene. However, metabolite profiles differed between rat and human homologues of cytochromes P450, in particular for cytochrome P450 1A2. The predominant metabolite formed by rat cytochrome P450 1A2 was the K region trans-9,10-dihydrodiol, whereas human cytochrome P450 1A2 produced similar amounts of the trans-1,2-, trans-3,4- and trans-9,10-dihydrodiol. High amounts of trans-1,2-dihydrodiol, the metabolic precursor of the bay-region dihydrodiol epoxide, were also formed by human cytochrome P450 1A1 compared to its rat homologue. Unexpectedly, human cytochrome P450 2E1 showed a remarkable catalytic activity to metabolize phenanthrene to its trans-9,10-dihydrodiol. Utilizing recombinant CYPs in live V79 cells appears to be a valuable too yielding results important for the evaluation of exposure data and risk assessment for humans.     

Metabolic activation of dibenzo[a,l]pyrene by human cytochrome P450 1A1 and P450 1B1 expressed in V79 Chinese hamster cells

Metabolic activation of the strongly carcinogenic polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P) and its trans-8,9-dihydrodiol (trans-8,9-diol) catalyzed by human cytochromes P450 (P450) 1A1 and 1B1 was investigated. DNA binding of DB[a,l]P in mammalian cell lines has previously been shown to be preferentially mediated by fjord region DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE). In order to elucidate different capabilities of both P450 enzymes for metabolic activation of DB[a, l]P V79 Chinese hamster cells, stably expressing human P450s 1A1 or 1B1 have been exposed to the parent PAH or its racemic trans-8, 9-diol. For this purpose, synthesis and spectroscopic characterization of the trans-DB[a,l]P-8,9-diol and its individual enantiomers have been achieved. Both human P450-expressing cell lines were capable of transforming DB[a,l]P to its fjord region DB[a, l]PDE, but the extent of metabolism to DB[a,l]PDE catalyzed by human P450 1B1 was higher compared to human P450 1A1 at all times measured. On the other hand, cytotoxicity studies performed with the same incubation systems emerged stronger effects by DB[a,l]P and its enantiomeric trans-11,12-diols in human P450 1A1-expressing cells. Both human P450 enzymes stereospecifically catalyzed the formation of the (-)-DB[a,l]P-11,12-diol with R,R-configuration, whereas only the human P450 1A1-expressing cells form small amounts of the K-region trans-8,9-diol with high excess of the (+)-(8R, 9R)-enantiomer. Application of trans-DB[a,l]P-8,9-diol in metabolism studies revealed that this compound is converted by human P450s 1A1 and 1B1 to several diol phenols and bis-diols. However, and even at concentrations as high as 10 microM, in both cell lines the trans-DB[a,l]P-8,9-diol showed no cytotoxicity at all, suggesting that an activation of DB[a,l]P via further oxidation of the K-region trans-8,9-diol plays a minor role.     

Genetically engineered bacterial cells co-expressing human cytochrome P450 with NADPH-cytochrome P450 reductase: prediction of metabolism and toxicity of drugs in humans

Genetically engineered bacterial cells expressing human cytochrome P450 (CYP) have been developed as new tools to predict the metabolism and toxicity of drugs in humans. There are various host cells for the heterologous expression of a form of CYP. Among them, bacterial cells such as Escherichia coli (E. coli) have advantages with regard to ease of use and high yield of protein. CYP protein could be first expressed by the modification of the N-terminal amino acid sequence in E. coli cells in 1991. Since then, many forms of human CYP have been successfully expressed in E. coli cells. Since the E. coli cells do not possess endogeneous electron transport systems to support the full catalytic activity of CYP, E. coli strains co-expressing both human CYP and NADPH-cytochrome P450 reductase (OR) have been established. Each form of CYP expressed in the E. coli cells efficiently catalyzed the oxidation of a representative substrate at an efficient rate, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. According to the studies performed so far, the modification of the N-terminal amino acid sequence of CYP did not seem to affect the catalytic properties of CYP. The human CYP expressed in the E. coli cells were applicable for studies to determine a metabolic pathway(s) of drugs and to estimate kinetic parameters of drug metabolism by human CYP. Drug-drug interactions caused by inhibition of the metabolism of drugs by human CYP could also be examined by in vitro inhibition studies with CYP expressed in the E. coli cells. Recently, human CYP was co-expressed with the OR in Salmonella typhimurium (S. typhimurium) cells used for mutation assay (Ames test) by applying the technology for the expression of human CYP and the OR in E. coli cells, to evaluate whether chemicals including drugs are metabolically activated by human CYP and show mutagenicity. These strains of bacteria are considered as useful tools to study the metabolism and the toxicity of drugs in humans.     

Regio- and stereoselectivity in the metabolism of benzo[c]phenanthrene mediated by genetically engineered V79 Chinese hamster cells expressing rat and human cytochromes P450

Regio- and stereoselective metabolism mediated by cytochrome P450 (CYP) and metabolite-dependent cytotoxicity of benzo[c]phenanthrene (B[c]Ph) and its trans-3,4-dihydrodiol, the metabolic precursor of the carcinogenic fjord-region B[c]Ph-3,4-dihydrodiol 1,2-epoxides (B[c]PhDE), were investigated with V79 Chinese hamster cells genetically engineered for three rat and six human CYP isoforms. The order of the capabilities of the CYP isoforms to metabolize B[c]Ph was as follows: h1A1>r1A1>r1A2>h1B1>h1A2>r2B1>h2E1>h2A6>h3A4. Regardless of the species, all individual CYP isoforms preferentially catalyzed the oxidation of B[c]Ph at the 5,6-position (K-region) except human CYP1A1 and human CYP1A2, which oxidized both the 5,6- and the 3,4-position with similar efficiency. While human CYP1A1, rat CYP1A1 and rat CYP1A2 formed almost exclusively the (-)-B[c]Ph-3R,4R-dihydrodiol, human CYP1A2 produced both the (-)-3R,4R- and the (+)-3S,4S-dihydrodiol enantiomers in a ratio of 2:1. Stereoselective activation of B[c]Ph, the (±)-B[c]Ph-3,4-dihydrodiol and its (-)-3R,4R-enantiomer to the fjord-region (-)-anti-B[c]PhDE occurred upon incubation with rat CYP1A1 and rat CYP1A2 as indicated by the formation of two stereoisomeric tetraols, the hydrolysis products of the labile anti-B[c]PhDE. The formation of tetraols in the culture medium was accompanied by a concentration-dependent increase in cytotoxicity indicating that this effect was mediated by the fjord-region (-)-anti-B[c]PhDE formed as reactive intermediate. All human and rat CYP-expressing V79 cell lines investigated did not show any significant capacity to metabolize the (+)-3S,4S-dihydrodiol. The present study indicates that the human CYP isoforms 1A1 and 1B1 have complementary catalytic properties to activate B[c]Ph to its fjord-region B[c]PhDE, whereas other human isoforms play a minor role. Activation of B[c]Ph by human CYP1A1 and 1B1 is less efficient than by rat CYP1A1 or rat CYP1A2, but proceeds with similar stereoselectivity via the (-)-3R,4R-dihydrodiol to the strong carcinogen (-)-anti-B[c]PhDE with (R,S,S,R)-configuration.     

V79 Chinese hamster cells express cytochrome P-450 activity after simultaneous exposure to polycyclic aromatic hydrocarbons and aminophylline

V79 Chinese hamster lung cells expressed low but significant aryl hydrocarbon hydroxylase activities when treated with an inducer of cytochrome P-450I, such as benz[alpha]anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), together with aminophylline. Inducibility by polycyclic aromatic hydrocarbons and inhibition by a specific monoclonal antibody indicated that the observed enzyme activity was mediated by cytochrome P-450I. Intact V79 cells pretreated with TCDD and aminophylline for 24 h metabolized benzo[alpha]pyrene to phenolic products which accumulated linearly in the growth medium for at least the same time period. Exposure of V79 cells to 10 microM benzo[alpha]pyrene and aminophylline for 72 h reduced subsequent cell growth by about 40%. The results demonstrate that V79 cells, under specific conditions, express PAH-inducible cytochrome P-450I and are capable of activating benzo[alpha]pyrene to cytotoxic products.     

The expression of human cytochrome P450IA1 in the yeast Saccharomyces cerevisiae

Data from animal studies suggest that cytochrome P450IA1 catalyses the metabolic activation of several procarcinogenic compounds. In the present study, we have expressed human cytochrome P450IA1 in yeast cells. A 1.70 kb BclI/BamHI fragment containing a full-length human cytochrome P450IA1 cDNA was inserted into the BglII expression site of the yeast expression plasmid pMA91 thereby allowing the ATG initiation codon to be located adjacent to the PGK (phosphoglycerate kinase) promoter. The resulting recombinant plasmid, pCK-1, was introduced into Saccharomyces cerevisiae strains ATCC 44773 and AH22. Microsomes prepared from yeast transformatants of strain ATCC 44773 contained undetectable levels of cytochrome P450. In contrast, microsomes from strain AH22 contained cytochrome P450 with a specific content of 33.3 +/- 10.8 pmol/mg of microsomal protein and showed a reduced carbon monoxide difference spectrum with a peak at 448 nm. Control yeast cells transformed with pMA91 showed no cytochrome P450. Western blots were carried out using an antibody that reacts against rat cytochrome P450IA1 and an antibody that reacts against a synthetic peptide representing a short sequence of human cytochrome P450IA1. A band with a molecular weight of 54 kD was observed in microsomes of yeast transformed with pCK-1, but not with pMA91. When microsomes from yeast transformed with pCK-1 were incubated with benzo(a)pyrene (10 min, 10-160 microM), an estimated Km value of 7 microM was obtained. The availability of yeast cells with functionally active human cytochrome P450IA1 will facilitate molecular structure-activity studies of procarcinogen and drug metabolism by this enzyme in man.     

Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes

Ellipticine is a potent antineoplastic agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). We examined a panel of genetically engineered V79 cell lines including the parental line V79MZ and recombinant cells expressing the human CYP enzymes CYP1A1, CYP1A2 or CYP3A4 for their ability to activate ellipticine. The extent of activation was determined by analysing DNA adducts by 32P-postlabelling. Ellipticine was found to be toxic to all V79 cell lines with IC(50) values ranging from 0.25 to 0.40 microM. The nuclease P1 version of the 32P-postlabelling assay yielded a similar pattern of ellipticine-DNA adducts with two major adducts in all cells, the formation of only one of which was dependent on CYP activity. This pattern is identical to that detected in DNA reacted with ellipticine and the reconstituted CYP enzyme system in vitro as confirmed by HPLC of the isolated adducts. Total adduct levels ranged from 2 to 337 adducts per 10(8) nucleotides, in the parental line and in V79 expressing CYP3A4, respectively. As in vitro, human CYP1A2 and CYP1A1 were less active. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in cells in culture, and confirm the formation of covalent DNA adducts as a new mechanism of ellipticine action.     

Effects of 2'-demethoxy-2'-propoxygriseofulvin on microtubule distribution in Chinese hamster V79 cells

Griseofulvin may combine with tubulin as a component of microtubules and, at high doses, inhibits microtubule formation in mammalian cells. In this study, the author examined the effects of three various 2'-substitutive derivatives of griseofulvin on microtubule distribution in Chinese hamster V79 cells. The results showed that 2'-demethoxy-2'propoxygriseofulvin was the strongest inhibitor of a normal cytoplasmic microtubule network, unlike findings in previous in-vitro studies.     

Interspecies features of hepatic cytochromes P450 IA1 and P450 IA2 in rodents

1. Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) both inhibit the O-deethylation of 7-ethoxyresorufin (ER) in liver microsomes of benzo(a)pyrene-induced (BP) mice but do not inhibit the O-deethylase activity in liver microsomes of BP-induced rats.

2. Anti-P3-450 and anti-P-450d-c inhibit BP hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this reaction at all in BP-induced rat liver microsomes.

3. Isolated cytochrome P3-450 in a reconstituted monooxygenase system metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, does not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min per nmol cytochrome.

4. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes corresponding to cytochrome P1-450, 55.0 kDa, and cytochrome P3-450, 54.5 kDa. There appeared a single band (cytochrome P3-450) in interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c.     

Interspecies features of hepatic cytochromes P450 IA1 and P450 IA2 in rodents

1. Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) both inhibit the O-deethylation of 7-ethoxy-resorufin (ER) in liver microsomes of benzo(a)pyrene-induced (BP) mice but do not inhibit the O-deethylase activity in liver microsomes of BP-induced rats. 2. Anti-P3-450 and anti-P-450d-c inhibit BP hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this rection at all in BP-induced rat liver microsomes. 3. Isolated cytochrome P3-450 in a reconstituted monooxygenase system metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, does not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min per nmol cytochrome. 4. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes corresponding to cytochrome P1-450, 55.0 kDa, and cytochrome P3-450, 54.5 kDa. There appeared a single band (cytochrome P3-450) in interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c.     

Toxicity of 3-nitronaphthalimides to V79 379A Chinese hamster cells

The cellular uptake and toxicity of a number of substituted 3-nitronaphthalimides was investigated. Uptake of these compounds into cells was initially rapid and reached a plateau after several hours, where in some cases intracellular concentrations were much greater than the corresponding extracellular concentrations. Little uptake was obtained, however, with a compound carrying an acidic substituent. Toxicity studies divided the compounds into two main groups; those where survival curves were convex and those where survival curves were concave. The shapes of survival curves of the latter group did not appear to reflect depletion of extracellular drug. Uptake and toxicity of different drugs were not well correlated and bioreductive metabolism of the nitro-substituent did not appear to be a major contributor to toxicity. There was no consistent differential toxicity of these drugs in aerobic and hypoxic conditions. It was concluded that the nature of the ring substituent had more effect on toxicity than the absolute concentration of the naphthalimide ring or bioreductive metabolism of the nitro-group.     

Inhibition of DNA replication by ozone in Chinese hamster V79 cells

DNA replication in Chinese hamster lung fibroblasts, line V79, was depressed in a dose-dependent manner over an ozone concentration range of 1-10 ppm. When the cells were exposed for 1 h at concentrations up to 6 ppm, the rate of DNA replication, as measured by [3H]thymidine incorporation, declined further during a 3-h period immediately following exposure. At higher ozone concentrations, at which more than 99.9% of the cells were killed, no further decline in DNA replication was seen beyond that immediately following exposure. Cultures exposed for 1 h to 10 mM ethyl methanesulfonate or to 10 J/m2 of ultraviolet (UV) light showed a similar progressive decline in the rate of DNA replication. The inhibition of DNA replication by ozone resembled that seen after exposure of cells to chemical mutagens or radiation and did not resemble the inhibition produced by metabolic poisons. The results may indicate that ozone or its reaction products interact directly with DNA in a way that inhibits replication.     

Nuclease hypersensitivity of the rat cytochrome P450IA1 gene

The bovine pancreatic deoxyribonuclease I (DNAase I) hypersensitivity of the rat cytochrome P450IA1 gene was investigated. A nuclease-hypersensitive region was observed at approximately 3.2 to 5.1 kilobase pairs upstream of exon 1 in adult and fetal rat liver. This region did not necessarily correlate with gene expression following 3-methylcholanthrene induction, although it may determine the potential for inducibility of this gene.     

Isolation of a bovine full length cytochrome P450 (CYP3A) cDNA sequence and its functional expression in V79 cells

From a bovine liver cDNA library in λMaxl a 1870 bp cDNA was isolated using the human CYP3A4 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 507 amino acids and exhibited homologies of 76, 72 and 64% with canine CYP3A12, human CYP3A4 and rat CYP3A1, respectively. Furthermore, a very high homology of 91.7% was observed with the deduced amino acid sequence of a partial CYP3A cDNA from dwarf goat. A striking observation was that both the bovine and the goat cDNA exhibit a 4 amino acid extension at the C-terminus, which is due to a frame-shifting insertion of 2 nt. The bovine CYP3A cDNA was cloned in a retroviral vector, transfected to V79 cells and cells were selected for cytochrome P450 expression. The expressed enzyme was shown to catalyze the 6β-hydroxylation of testosterone, which could also be observed in a V79 cell line expressing human CYP3A4. In the bovine CYP3A cell line, however, 6β-hydroxytestosterone was not found to be the major metabolite. This cell line additionally showed high levels of hydroxylase activity at the 2β and 12β position of testosterone. The cDNA-expressed testosterone hydroxylase activity could be inhibited with the specific CYP3A inhibitors, tiamulin and ketoconazole.     

Development of a V79 cell line expressing human cytochrome P450 2D6 and its application as a metabolic screening tool

Expression of human cytochrome P450 (CYP) in heterologous cells is a means of specifically studying the role of these enzymes in drug metabolism. The complete cDNA encoding CYP2D6-VAL(374) was inserted into an expression vector containing the strong mycloproliferative sarcoma virus promotor in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP2D6 cDNA was confirmed by polymerase chain reaction analysis. The protein expression was shown by Western blotting. Functional expression could be demonstrated by O-demethylation of dextromethorphan to dextrorphan in live cells. The enzymatic activity of 154 ± 16 pmol min(-1) mg(-1) protein was comparable with dextromethorphan-O-demethylation activities of human liver. The metabolism of two dopaminergic ergoline derivatives was investigated in whole recombinant V19 cells. Both lisuride and terguride were monodeethylated; in case of lisuride a correlation to the in vivo situation was demonstrated comparing poor and extensive metabolizers.