The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability > or = 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is > or = 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.
Dibenzo[a,l]pyrene (DB[a,l]P) is an environmental contaminant and a very potent carcinogen. DB[a,l]P exceeds the carcinogenic potency of both benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in rodent bioassays. Previous studies demonstrated that DB[a,l]P is metabolized to DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE) in the human mammary carcinoma cell line MCF-7. In the present study the major DNA adducts formed in DB[a,l]P-treated MCF-7 cells have been identified through the use of 33P-postlabeling. TLC and HPLC. DB[a,l]P is metabolically activated in MCF-7 cells to form large amounts of three major DNA adducts and smaller amounts of three other adducts. The three major DNA adducts are with deoxyadenosine: two are formed by reaction of (+)-syn-DB[a,l]PDE (11S,12R,13S,14R), the third by reaction of (-)-anti-DB[a,l]PDE (11R,12S,13S,14R). The results demonstrate that DB[a,l] is stereoselectively metabolized in MCF-7 cells to form one enantiomer of each diol epoxide diastereomer; (+)-syn-DB[a,l]PDE and (-)-anti-DB[a,l]PDE. The high extent of binding of these diol epoxides to deoxyadenosine in DNA of MCF-7 cells may help to explain the very high carcinogenic potency of DB[a,l]P and suggests that DB[a,l]P could also pose a carcinogenic threat to humans.
Extensive tumorigenicity studies in rodents revealed that dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen among all polycyclic aromatic hydrocarbons (PAHs) tested so far. The structure of the genotoxic metabolite(s) responsible for this exceptional carcinogenicity is unknown. The fjord-region syn- and anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (syn- and anti-DB[a,l]PDE) were synthesized to clarify their role as possible ultimate mutagenic and carcinogenic metabolites of DB[a,l]P.9-Formyl-11,12-dimethoxybenzo[g] chrysene was prepared from 9-phenanthrylacetic acid by a photochemical route. After reaction of the aldehyde with trimethylsulfonium iodide to generate an oxiranyl side-chain, treatment with boron trifluoride produced the key intermediate 11,12-dimethoxy-DB[a,l]P in 14% overall yield. From 11,12-dimethoxy-DB[a,l]P the syn- and anti-DB[a,l]PDE were stereoselectively prepared via the trans-11,12-dihydrodiol. The mutagenicity of the syn- and anti-DB[a,l]PDE was examined in four his- strains of Salmonella typhimurium and in Chinese hamster V79 cells. In all five test systems, the new dihydrodiolepoxides were more potent than any of the previously investigated dihydrodiolepoxides. The specific mutagenicity observed with anti-DB[a,l]PDE in strain TA104 exhibited the highest value ever found with any compound in any his- strains of S.typhimurium. The same appears to be true for the activity observed with this compound in V79 cells. In all five systems, syn-DB[a,l]PDE was only moderately less active than its anti-diastereomer (approximately 2-fold). The exceptional mutagenic activities of these dihydrodiolepoxides may be one of the reasons for the exceptional carcinogenic activity of DB[a,l]P.
being recrystallized from CHC13/CH30H/C8H8 to give 8: 0.12 g (25.9%); mp 194-195 °C; NMR (CD3CN/D20) 8 3.2 (m, 2
Thirteen compounds, isolated from spinach (Spinacia oleracea), acted as antimutagens against the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in Salmonella typhimurium TA 98. The antimutagens were purified by preparative and micropreparative HPLC from a methanol/water (70:30, v/v) extract of dry spinach (commercial product) after removal of lipophilic compounds such as chlorophylls and carotenoids by solid-phase extraction (SPE). Pure active compounds were identified by instrumental analysis including FT-IR, (1)H and (13)C NMR, UV-vis spectroscopy, and mass spectrometry. All of these compounds were flavonoids and related compounds that could be attributed to five groups: (A, methylenedioxyflavonol glucuronides) 5,3'-dihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 1), 5,2',3'-trihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 2), 5-hydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 3); (B, flavonol glucuronides) 5,6,3'-trihydroxy-7,4'-dimethoxyflavonol 3-O-beta-glucuronide (compound 4), 5,6-dihydroxy-7,3',4'-trimethoxyflavonol 3-O-beta-glucuronide (compound 5); (C, flavonol disaccharides) 5,6,4'-trihydroxy-7,3'-dimethoxyflavonol 3-O-disaccharide (compound 6), 5,6,3',4'-tetrahydroxy-7-methoxyflavonol 3-O-disaccharide (compounds 7 and 8); (D, flavanones) 5,8,4'-trihydroxyflavanone (compound 9), 7,8,4'-trihydroxyflavanone (compound 10); (E, flavonoid-related compounds) compounds 11, 12, and 13 with incompletely elucidated structures. The yield of compound 1 was 0.3%, related to dry weight, whereas the yields of compounds 2-13 ranged between 0.017 and 0.069%. IC(50) values (antimutagenic potencies) of the flavonol glucuronides ranged between 24.2 and 58.2 microM, whereas the flavonol disaccharides (compounds 7 and 8), the flavanones (compounds 9 and 10), and the flavonoid-related glycosidic compounds 11-13 were only weakly active. The aglycons of compounds 7 and 8, however, were potent antimutagens (IC(50) = 10.4 and 13.0 microM, respectively).
Dibenzo[a,l]pyrene (DB[a,l]P) represents the most potent carcinogenic polycyclic aromatic hydrocarbon (PAH) yet discovered. Like other PAHs, DB[a,l]P requires metabolic activation to exert its mutagenic and/or carcinogenic activity. In the human mammary carcinoma cell line MCF-7, DB[a,l]P is stereoselectively metabolized to the (-)-anti- and (+)-syn-DB[a,l]P-11,12-diol 13,14-epoxides (DB[a,l]PDE) which both bind extensively to deoxyadenosine residues in DNA. To further characterize the underlying mechanism of its strong carcinogenicity, the relationship between DNA binding and mutagenicity of DB[a,l]P was determined. Racemic DB[a,l]P-11,12-dihydrodiol and the two individual (+)- and (-)-enantiomers, the metabolic precursors of the stereoisomeric fjord region dihydrodiol epoxides, were also investigated. Induction of mutations at the HPRT locus was measured in a MCF-7 cell-mediated Chinese hamster V79 cell mutation assay. The parent hydrocarbon, (+/-)-DB[a,l]P-11,12-dihydrodiol, and (-)-DB[a,l]P-11,12-dihydrodiol were highly mutagenic under the assay conditions. In contrast, (+)-DB[a,l]P-(11S,12S)-dihydrodiol was not mutagenic using MCF-7 cells as the metabolic activating system. Analysis of DNA adducts in the same experiments revealed that MCF-7 cells treated with (-)-DB[a,l]P-11,12-dihydrodiol formed exclusively (-)-anti-DB[a,l]-PDE adducts whereas cells treated with (+)-DB[a,l]P-11,12-dihydrodiol did not contain detectable levels of DNA adducts. These results suggest that specific cytochrome P450 enzymes may have high stereoselectivity for activation of the two DB[a,l]P-11,12-dihydrodiol enantiomers, and this may play an important role in the metabolic activation of the strong carcinogen DB[a,l]P in human cells.
The nature of stable DNA adducts derived from the very potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in the presence of rat liver microsomes in vitro and in mouse skin in vivo has been studied using 32P-postlabeling and laser-based fluorescence techniques. Analysis of DB[a,l]P-DNA adducts via 32P-postlabeling has been obtained by comparison of the adduct patterns to those obtained from reactions of synthetic (+/-)-anti-, (+)-anti-, (-)-anti-, and (+/-)-syn-DB[a,l]P-11,12-diol 13,14-epoxide (DB[a,l]PDE) with single nucleotides and calf thymus DNA. anti-DB[a,l]PDE-dA adducts derived from the (-)-enantiomer are the major adducts formed in calf thymus DNA and in mouse skin DNA. The ratio of deoxyadenosine to deoxyguanosine modification is approximately 2:1 in mouse skin exposed to DB[a,l]P; activation by rat liver microsomes leads to a similar profile of adducts but with two additional spots. The conformations of DB[a,l]P adducts in native DNA, as well as the possibility of conformation-dependent repair, have been explored by low-temperature fluorescence spectroscopy. These studies have been performed using polynucleotides and calf thymus DNA reacted in vitro with DB[a,l]PDE and native DNA from mouse epidermis exposed to DB[a, l]P. The results show that adducts are heterogeneous, possess different structures, and adopt different conformations. External, external but base-stacked and intercalated adduct conformations are observed in calf thymus DNA and in mouse skin DNA samples. Differences in adduct repair rates are also revealed; namely, the analysis of mouse skin DNA samples obtained at 24 and 48 h after exposure to DB[a,l]P clearly shows that external adducts are repaired more efficiently than intercalated adducts. These results, taken together with those for B[a]P-DNA adducts [Suh et al. (1995) Carcinogenesis 16, 2561-2569], indicate that the repair of DNA damage resulting from PAH diol epoxides is conformation-dependent.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.