No abstract

Alkyl-substituted polycyclic aromatic hydrocarbons may be metabolized to highly reactive benzylic sulfuric acid esters via benzylic hydroxylation and subsequent sulfonation. We have studied the benzylic hydroxylation of 1-methylpyrene (MP), a hepatocarcinogen in rodents, and 1-ethylpyrene (EP), whose benzylic hydroxylation would produce a secondary alcohol (alpha-HEP), in contrast to the primary alcohol (alpha-HMP) formed from MP. The hydrocarbons were incubated with hepatic microsomal preparations from humans and rats, as well as with V79-derived cell lines engineered for the expression of individual cytochrome P450 (CYP) forms from human (1A1, 1A2, 1B1, 2A6, 2E1, 3A4) and rat (1A1, 1A2, 2B1). All microsomal systems and CYP-expressing cell lines used, but not CYP-deficient V79 cells, showed biotransformation of both hydrocarbons. Formation of the benzylic alcohol was detected in each case. alpha-HMP and its oxidation product, 1-pyrenylcarboxylic acid (COOH-P), accounted for a major part of the total amount of the metabolites formed from MP in the presence of human liver microsomes (38-64%) and cells expressing human 3A4, 2E1 or 1B1 (80-85%). Likewise, cells expressing human 1A1 showed a higher contribution of alpha-HMP and COOH-P to the total metabolites (45%) than cells expressing the orthologous enzyme of the rat (3%). EP was metabolized at a higher rate and with modified regioselectivity compared with MP, although omega-hydroxylation of the side chain was not detected with the cell lines and only accounted for a small percent of the biotransformation by the microsomal preparations. The highest contributions of alpha-HEP to the total metabolites from EP were detected with the cells expressing human 1A1, 1B1 and 3A4 (38-51%). alpha-HEP accounted for 16% of the metabolites formed in the presence of human hepatic microsomes. Thus, benzylic hydroxylation is a major initial step in the metabolism of MP and EP. This pathway appears to be even more important in humans than in rats. Previously, we had shown that the second step of the activation, the sulfonation of alpha-HMP and alpha-HEP, is also efficiently catalysed by various forms of human sulfotransferases.

Tamoxifen increases the risk of human endometrial cancer and is a potent carcinogen in rat liver, in which it produces DNA adducts and cytogenetic damage. Nevertheless its prophylactic use against breast cancer in healthy women is under investigation in several large trials. To investigate whether rat hepatocarcinogenicity predicts human hepatocarcinogenicity we used genetically engineered bacterial and mammalian target cells to investigate how α-hydroxytamoxifen, a major phase I metabolite of tamoxifen, is further metabolised by rat and human phase II enzymes, sulfotransferases, to mutagenic and DNA-adduct-forming species. We expressed rat hydroxysteroid sulfotransferase a, a liver-specific enzyme, and corresponding human sulfotransferase in bacteria (Salmonella typhimurium) and in a mammalian cell line (Chinese hamster V79 cells) and tested α-hydroxytamoxifen for DNA adduct formation and mutagenicity in these systems, using unmodified cells as controls. In cells that expressed rat hydroxysteroid sulfotransferase, α-hydroxytamoxifen was mutagenic and formed the same pattern of DNA adducts as that found in the liver of tamoxifen-treated rats. α-Hydroxytamoxifen was not activated, or was at least 20 times less active in cells expressing human hydroxysteroid sulfotransferase. All the other six known human xenobiotic-metabolising sulfotransferases were also expressed in S.typhimurium. None activated α-hydroxytamoxifen to a mutagen. These results suggest that the risk of DNA adduct formation, and cancer, in the human liver is low and explain why tamoxifen is a powerful carcinogen to the rat liver, and why standard short-term tests fail to detect its mutagenicity.

Biosynthesis of polygalacturonase (PG) by A. niger strain R 1/214 correlates with the morphology of mycelium in submerged culture. The mean specific PG-synthesis (PG-U.g-1.h-1) increases with the degree of compactness of mycelium. PG-production can be optimized by a precise adjustment of the culture conditions after direct spore inoculation (diffuse mycelium) but the high synthesis as by compact mycelium is never obtained. Different reasons for the higher enzyme production by the pellet mycelium are discussed. PG-synthesis is assumed to be strictly connected with a limitation of nutrient and oxygen supply.

A bioassay procedures utilising the western-banded blow fly, Chrysomya albiceps (Diptera-Calliphoridae) has been used to guide the fkactionation of an ethanolic extract from the leaves of Nerium oleander (Apocynaceae). The cardiotonic glycoside, neriifolin: (3-[(6-deoxy-3-0-methyl-a-L glucopyranosyl) oxyl-14-hydroxy-5 p Card-20 [22]-endolide) was crystallised from the insecticidal active fraction of the ethanolic extract. The values of LC,, of the ethanolic extract, active fraction, isolated crystals and authentic neriifolin were 164 ppm, 57 ppm, 35 ppm and 36 ppm, respectively when incorporated into the blow fly diet. A primary test on the cytotoxicity and mutagenicity of the crude extract against non-target organisms was achieved by determining the cytotoxicity and mutagenicity on a mammalian cell line using different concentrations of the extract (in vitro) through a radioactive thymidine incorporation technique and hypoxanthine phosphoribosyl transferase (HPRT) test, using the Chinese hamster lung fibroblasts (V79-MZ cell line). Cytotoxicity tests revealed that the LC,, was approximately 200 ppm, and the mutagenicity was very low compared to the standard active mutagen.