Benzylic hydroxylation of 1-methylpyrene and 1-ethylpyrene by human and rat cytochromes P450 individually expressed in V79 Chinese hamster cells.

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.

Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors.

To discover safe and effective topical skin-lightening agents, we have evaluated alkyl esters of the natural product gentisic acid (GA), which is related to our lead compound methyl gentisate (MG), and four putative tyrosinase inhibitors, utilizing mammalian melanocyte cell cultures and cell-free extracts. Desirable characteristics include the ability to inhibit melanogenesis in cells (IC50 < 100 microg/mL) without cytotoxicity, preferably due to tyrosinase inhibition. Of the six esters synthesized, the smaller esters (e.g. methyl and ethyl) were more effective enzyme inhibitors (IC50 approximately 11 and 20 microg/mL, respectively). For comparison, hydroquinone (HQ), a commercial skin "bleaching" agent, was a less effective enzyme inhibitor (IC50 approximately 72 microg/mL), and was highly cytotoxic to melanocytes in vitro at concentrations substantially lower than the IC50 for enzymatic inhibition. Kojic acid was a potent inhibitor of the mammalian enzyme (IC50 approximately 6 microg/mL), but did not reduce pigmentation in cells. Both arbutin and magnesium ascorbyl phosphate were ineffective in the cell-free and cell-based assays. MG at 100 microg/mL exhibited a minimal inhibitory effect on DHICA oxidase (TRP 1) and no effect on DOPAchrome tautomerase (TRP-2), suggesting that MG inhibits melanogenesis primarily via tyrosinase inhibition. MG and GA were non-mutagenic at the hprt locus in V79 Chinese hamster cells, whereas HQ was highly mutagenic and cytotoxic. The properties of MG in vitro, including (1) pigmentation inhibition in melanocytes, (2) tyrosinase inhibition and selectivity, (3) reduced cytotoxicity relative to HQ, and (4) lack of mutagenic potential in mammalian cells, establish MG as a superior candidate skin-lightening agent.

Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture.

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 alpha-hydroxy-tamoxifen, 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 alpha-hydroxytamoxifen for DNA adduct formation and mutagenicity in these systems, using unmodified cells as controls. In cells that expressed rat hydroxysteroid sulfotransferase, alpha-hydroxytamoxifen was mutagenic and formed the same pattern of DNA adducts as that found in the liver of tamoxifen-treated rats. Alpha-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 alpha-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.

Cell culture system for the controlled intracellular generation of reactive oxygen species.

The LC(50) of duroquinone was about 25 times lower in a Chinese hamster V79-derived cell line which was genetically engineered for the expression of human cytochrome P-450 reductase (V79-MZ-hOR) than in the parental V79-MZ cell line. Reduction of the O(2) concentration in the atmosphere from 21 to 5% decreased the cytotoxicity of duroquinone by a factor of 4. The addition of duroquinone to the homogenate of V79-MZ-hOR cells led to the formation of Superoxide radicals, as demonstrated by the formation of formazan from nitroblue tetrazolium, and inhibition of this reaction in the presence of Superoxide dismutase. Taken together, these results indicate that the cytotoxicity of duroquinone in V79-MZ-hOR cells is caused by Superoxide [and/or other reactive oxygen species (ROS) formed from Superoxide], In this system, ROS can be formed continuously in a controlled manner within the indicator cells (e.g. by varying the concentrations of O(2) and duroquinone), and the parental V79-MZ cell line can be used as a control.

Influence of culture conditions on mycelial structure and polygalacturonase synthesis of Aspergillus niger.

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.

[Quantitative morphologic studies on hypoxically cultured rat myocardial cells]. / Quantitative morphologische Untersuchungen an hypoxisch kultivierten Rattenherzmuskelzellen

Ventricle cells from neonatal rats were cultured in a medium preventing cell proliferation on a modified Roller apparatus with a defined pericellular oxygen partial pressure (pO2) of 38 or 0.6 mm Hg for about one week. The cells were harvested after the second and eighth day of culture (corresponding to one or seven days of culture under a defined pO2) and prepared for electron microscopy. Electron micrographs of this material were examined by stereologic techniques. The obtained results showed some deviations of mitochondrial fine structure of heart muscle cells depending on oxygen supply. During cultivation with a pO2 of 0.6 mm Hg (hypoxic conditions) we observed a proportional increase in mitochondria without cristae and an increase in size accompanied by a more irregular shape. On the contrary, the mitochondria of cells cultured with a pericellular pO2 of 28 mm Hg, which we consider to be a normoxic condition, did not show any deviation of inner membrane arrangement, but an increase in number and a decrease of size during cultivation was apparent. The mitochondria-myofibrils ratio decreased during cultivation in both conditions of oxygen supply. The ratio between sarcoplasmatic reticulum and myofibrils decreased markedly at a pO2 of 0.6 mm Hg.