François Périn

Sexual differences in the expression of γ-glutamyl transpeptidase during 5,9-dimethyldibenzo[c,g]carbazole-induced hepatocarcinogenesis in mice

5,9-Dimethyl-7H-dibenzo[c,g]carbazole (5,9-diMeDBC) is an organ-specific carcinogen for mouse liver. It induces malignant hepatomas in 100% of XVIInc/Z and C57BL mice with a cumulative dose of only 12 mumol administered subcutaneously. Large sex-related differences in sensitivity towards this carcinogen were observed: entire males were much less sensitive than females. Castration of males nearly completely restored the sensitivity observed for females. The kinetics of appearance of gamma-glutamyl transpeptidase (gamma GT)-positive foci was enhanced in females and castrated males and was correlated with the tumour outbreak. On the contrary the expression of gamma GT in males was very reduced. The sex-dependent sensitivity seems to be regulated by a complex interplay of endogenous factors.

In vitro metabolism of N-methyl-dibenzo [c,g]carbazole a potent sarcomatogen devoid of hepatotoxic and hepatocarcinogenic properties

The metabolism of N-methyl substituted 7H-dibenzo[c,g]carbazole (N-Me DBC) was investigated in vitro using liver microsomes from 3-methylcholanthrene (MC)-, benzo[c]carbazole (BC) and Arochlor-pretreated mice and rats. N-Me DBC is a potent sarcomatogen devoid of hepatotoxicity and liver carcinogenic activity. The ethyl acetate-extractable metabolites were separated by high performance liquid chromatography (HPLC) and most of them were identified by proton magnetic resonance (PMR), mass spectrometry (MS) and comparison with synthetically prepared specimens. Mouse and rat microsomes gave rise to the same metabolites. The major metabolites were 5-OH-N-Me DBC (50%), N-hydroxymethyl (HMe) DBC (25-30%) and 3-OH-N-Me DBC (10%). Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO) to the standard incubation medium permitted the identification of two dihydrodiols among the minor metabolites. No metabolite of DBC was observed after incubation of N-Me DBC, or its major metabolite N-HMe DBC, with either mouse or rat microsomes, but the possibility of a slight demethylation cannot be totally excluded. The lack of biotransformation at the nitrogen atom site may explain the lack of hepatotoxicity and liver carcinogenic activity of N-Me DBC. The modulation of metabolism by epoxide hydrolase, cytosol and glutathione was also investigated. The results are discussed in the light of data previously obtained with hepatotoxic and hepatocarcinogenic DBC.

Morphological and carbogen-based functional MRI of a chemically induced liver tumor model in mice

A multifocal mouse liver tumor model chemically induced with 5,9‐dimethyl‐7H‐dibenzo[c,g]carbazole was investigated by respiratory‐triggered morphological and functional MRI (fMRI) at 4.7 Tesla. The model is characterized by the presence of two tumor types: hypovascular cholangioma and vascularized hepatocellular carcinoma (HCC). Growth curves measured by 3D‐MRI showed limited growth of cholangiomas and rapid growth of HCCs after a latency of about 25 weeks. Functional imaging based on T  2* ‐weighted fast gradient‐echo MRI and carbogen breathing was optimized for liver imaging in mice. A response to carbogen was observed in HCCs but not in cholangiomas. Transversal analysis (50 HCCs) of signal change upon carbogen revealed four different types of response patterns: 1) signal increase upon carbogen administration (74%); 2) small or insignificant signal change (10%), 3) transient signal decrease and delayed increase (8%), and 4) signal decrease (8%). Longitudinal follow‐up of a subgroup (N = 17) showed that an initially observed type 1 response, attesting to the presence of a functional vasculature, remained stable for at least 3 weeks in 14 HCCs. A switch from a type 1 response to another response type may be useful for demonstrating, in a noninvasive manner, a disturbance of tumor vasculature induced by anti‐vascular or anti‐angiogenic therapy. Magn Reson Med 50:522–530, 2003.

Structure and carcinogenicity of Dibenzo(c,g)carbazole derivatives

The carcinogenicity of several groups of carcinogens is evoked with particular reference to Dibenzo(c,g)carbazole derivatives. The activity of these derivatives is discussed with respect to their species and organ specificity. The enzymatic equipment is decisive as to whether the compounds formed can react with DNA or are simply detoxified and eliminated. All these carcinogens are complete carcinogens, i. e. they have the property of both initiation and promotion.

Comparative mutagenicity of 7H-dibenzo[c,g]carbazole and two derivatives in Muta™Mouse liver and skin

7H-Dibenzo[c,g]carbazole (DBC) is an environmental pollutant that produces DNA adducts and tumors in mouse liver and skin following subcutaneous injection and topical application. The two synthetic derivatives 5,9-dimethyl-DBC (DMDBC) and N7-methyl-DBC (NMDBC) induce tissue-specific lesions. DNA adducts and tumors are observed only in liver following exposure to DMDBC and only in skin following exposure to NMDBC. We used the positive selection MutaMouse model to measure the induction of mutations in the two target organs, 28 days after a single subcutaneous injection or topical application of DBC, DMDBC and NMDBC. In liver, DBC and DMDBC induced 30- to 50-fold increases in mutant frequency (MF), while NMDBC had only a weak effect, regardless of the route of administration. After topical application, DBC and NMDBC produced 3.4- to 7.9-fold increases in MF in skin, while DMDBC had a weak effect. After subcutaneous injection, the three compounds had no or weak effect in skin. This study shows gene mutations arise in the respective target organs in which primary DNA damage and tumors are observed. These results illustrate the relevance of the MutaMouse model for testing organ-specific mutagens.

Tissue‐specific differences in adduct formation by hepatocarcinogenic and sarcomatogenic derivatives of 7H‐dibenzo[c,g]carbazole in mouse parenchymal and nonparenchymal liver cells

Parenchymal (PC) and nonparenchymal (NPC) liver cells have different tissue‐specific, procarcinogen activation enzymes. NPC appear to be protected against the mutagenic effects of lipotropic bulky adducts induced by carcinogens by a unknown mechanism. Most studies of activation have been conducted with whole liver. The purpose of this study was to differentiate adduct formation in mouse PC and in NPC, isolated after in vivo administration of 7H‐dibenzo[c,g]carbazole (DBC), the most efficient liver carcinogen in mice, which also has potent sarcomagenic and epitheliomagenic activities. The very sensitive 32P‐postlabeling method was used to detect adducts. Two tissue‐specific DBC derivatives, 6‐methoxy‐DBC (6MeODBC), which is exclusively sarcomagenic, and 5,9‐dimethyl‐DBC (DiMeDBC), which is exclusively hepatocarcinogenic, were analyzed in parallel. Both PC and NPC generated the ultimate metabolites of DBC, but NPC were substantially less efficient. Clear‐cut tissue‐specific differences in adduct formation were established: the sarcomagenic 6MeODBC gave rise only to NPC‐DNA adducts, and the hepatocarcinogenic DiMeDBC only to PC‐DNA adducts. The chromatograms of the adducts were compared with those of mouse embryonic cells in culture and mouse epidermal cells. The results are discussed in connection with animal experiments with DBC, 6MeODBC, and dimethylbenzanthracene and with published data on PC and NPC activating enzymes. Environ. Mol. Mutagen. 29:346–356, 1997.

Organ-specific, carcinogenic dibenzo[c, g]carbazole derivatives: discriminative response in S. typhimurium TA100 mutagenesis modulated by subcellular fractions of mouse liver

7H-Dibenzo[c,g]carbazole (DBC) has carcinogenic effects on mouse subcutaneous fibroblasts and liver; the N-methyl derivative (N-MeDBC) induces only sarcomas; 3-methyl- and 5,9-dimethyldibenzo[c,g]carbazole (3-MeDBC and 5,9-DMeDBC) are specific, potent hepatocarcinogens in mice. The mutagenicity in S. typhimurium TA100 of these 4 compounds was evaluated in relation to the concentration of mouse liver 9000 X g supernatant (S9) and to the proportions of microsomes and cytosol in the medium. Optimal mutagenicity of N-MeDBC was seen with a low concentration of S9 or microsomes; a 5-10 times higher concentration of the subcellular fraction was necessary to induce optimal mutagenicity of the hepatocarcinogens 3-MeDBC and 5,9-DMeDBC. Intermediate quantities were needed in the case of DBC, which is carcinogenic in both cell types. Whereas the presence of cytosol had an inhibitory effect on the mutagenicity of the sarcomagenic N-MeDBC, the cytosolic fraction was essential for optimal mutagenic expression by the 2 hepatocarcinogenic derivatives. The activating cytosolic fraction is not inducible. These experiments show that programmed modulation of the metabolic activation system in the Ames test can be used to study organ-specific chemical carcinogenesis. The results suggest that differences in the enzymatic composition of target tissues are a determining factor in the organ specificity of carcinogens such as DBC.

ISOLATION AND PARTIAL CHARACTERIZATION OF A CYTOCHROME P-450 ISOENZYME (CYTOCHROME P-450TU) FROM MOUSE LIVER TUMORS

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.

Photobiological activities of 1,6-dioxapyrene in pro- and eukaryotic cells

The photobiological effect of a new pyrene derivative, 1,6-dioxapyrene (1,6-DP), was studied in Salmonella typhimurium (strain TA100) and in the diploid strain D7 of the yeast Saccharomyces cerevisiae. In Salmonella, 1,6-DP shows little mutagenicity in the dark in comparison to benzo[a]pyrene (B[a]P). This mutagenic activity decreases in the presence of liver S9 homogenates from Aroclor induced XVIInc/Z mice. However, in combination with 365 nm (UVA) radiation and in the absence of S9 mix, 1,6-DP behaves as an effective photodynamic compound inducing lethal and mutagenic effects in both organisms. In yeast, its activity, like that of B[a]P, is highly dependent on the presence of oxygen. For the same incident dose of UVA, 1,6-DP is, however, at least 6 times more effective than B[a]P in inducing cytotoxic and mutagenic effects. At equitoxic doses, 1,6-DP is as photomutagenic as B[a]P, suggesting that in both cases mutagenicity is due to similar mechanisms. Spectrophotometric measurements indicate physical interaction of 1,6-DP with DNA in the dark. Laser flash photolysis experiments show that 1,6-DP generates singlet oxygen with a quantum yield of 0.17. In vitro 1,6-DP produces oxidative damage to guanine bases specific for singlet oxygen mediated reactions. Alkaline step elution analysis of 1,6-DP plus UVA treated yeast cells indicates a decrease in average molecular weights in DNA and an induction of single strand breaks (ssb) originating from alkali labile sites. This effect is enhanced by D2O and is thus likely to be due to the production of singlet oxygen. The strand breaks appear to differ from those induced by gamma-rays because little, if any, repair of these ssb occurs during 30 min of post-treatment incubation in complete growth medium. These results suggest that the photobiological effects of 1,6-DP are due to oxidative damage in DNA mostly induced by singlet oxygen.

DNA adduct formation in primary mouse embryo cells induced by 7H-dibenzo[c,g]carbazole and its organ-specific carcinogenic derivatives

The nuclease P1 modification of the 32P‐postlabeling technique was used to study the biological activity of 7H‐dibenzo[c,g]carbazole (DBC) and some of its derivatives, including N‐methyldibenzo[c,g]carbazole (N‐MeDBC), 5,9‐dimethyldibenzo[c,g]carbazole (5,9‐diMeDBC), 5,9, N‐trimethyldibenzo[c,g] carbazole (5, 9, N‐triMeDBC), 6‐methoxydibenzo[c, g]carbazole (6‐McODBC), N‐acetyldibenzo[c,g]carbazole (N‐AcDBC), N‐hydroxymethyldibenzo[c,g]carbazole (N‐HMeDBC) in primary mouse embryo cells. A very good correlation was found between carcinogenic specificity in vivo of these N‐heterocyclic aromatic hydrocarbons and their DNA‐adduction in vitro. Primary mouse embryo cells were able to metabolize and detect tissue‐specific sarcomagens N‐MeDBC and 6‐MeODBC as well as derivatives with both sarcomagenic and hepatocarcinogenic activity, DBC, N‐AcDBC, and N‐HMeDBC. The strong specific hepatocarcinogen 5,9‐diMeDBC in vivo, did not induce any DNA‐adducts in the embryo cells, which suggests that the enzymatic composition of the target tissue probobly is the determining factor in the organ specificity of this derivative. 5,9,N‐triMeDBC, derivative without any carcinogenic activity in vivo, did not induce any DNA‐adducts in primary mouse embryo cells. Pretreatment of cells with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) apparently stimulated DNA‐adduct formation in the cells exposed to DBC, 6‐MeODBC, and N‐MeDBC. No or a very slight effect of TCDD on DNA‐adduct formation was found in cells exposed to N‐HMeDBC and N‐AcDBC. Preliminary results have shown that TCDD slightly induced cytochrome P4501A1 linked ethoxyresorufin O‐deethylase (EROD) activity in primary mouse embryo cells. These data suggest the role of cytochrome P4501A1 in the metabolism of DBC derivatives with sarcomagenic activity. Environ. Mal. Mutagen. 30:56–64, 1997