Genotoxicity of carcinogenic N-nitrosopropylamine derivatives in the hepatocyte primary culture/DNA-repair test

The genotoxicity of N-nitrosodipropylamine, 8 of its oxidized derivatives and N-nitroso-2,6-dimethylmorpholine was examined in the hepatocyte primary culture (HPC)/DNA repair test. Nine N-nitrosamines which are known to be carcinogenic and mutagenic were clearly positive in the HPC/DNA-repair test. N-Nitroso(2,3-dihydroxypropyl) (2-hydroxypropyl)amine did not elicit DNA repair, but showed a borderline mutagenic response in the Salmonella/microsome test. Thus, the HPC/DNA-repair test displays a comparable capacity to the bacterial mutagenesis test for detecting the genotoxic effects of this class of carcinogens.     

Genotoxicity of epoxy resin hardeners in the hepatocyte primary culture/DNA repair test

The genotoxicity of 9 chemicals used as epoxy resin hardeners was examined in the DNA repair test with rat hepatocytes. DNA repair synthesis was elicited by 7 chemicals, i.e., 4-aminodiphenyl ether, 4,4-diaminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether, 1,3-phenylenedi-4-aminophenyl ether, 4,4′-diaminodiphenyl methane and 4,4′-methylene-bis(2-chloroaniline).The positive results obtained with 4 epoxy resin hardeners of unknown carcinogenicity, i.e., 4-aminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether and 1,3-phenylene-di-4-aminophenyl ether suggest that they may be carcinogens. The genotoxicity of 1,4-phenylene-di-4-aminophenyl ether, of unknown carcinogenicity, and 4,4′-diaminodiphenyl sulfone, for which there is no sound proof of carcinogenicity, was not confirmed in the DNA repair test. The result with 4,4′-diaminodiphenyl sulfone was in agreement with its lack of mutagenicity in Salmonella typhimurium.     

Genotoxicity of pyrene oxide and 1-nitropyrene oxides in hepatocyte primary culture/DNA repair test

The genotoxicity of a pyrene oxide, 1-nitropyrene (NP) oxides and other related compounds was examined in the hepatocyte primary culture (HPC)/DNA repair test. Pyrene 4,5-oxide and both 1-NP-4,5-oxide and 1-NP-9,10-oxide elicited clearly positive responses of DNA repair. In this assay, 1-NP itself was weakly positive. However, other related chemicals such as pyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6-hydroxypyrene, and 1-nitro-8-hydroxypyrene did not generate positive responses.     

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes I. Mutagenicity in Salmonella typhimurium

Ptaquiloside, a potent carcinogen of an illudane-type sesquiterpene glycoside isolated from Pteridium aquilium, and its related compounds, hypolosides having the same nucleus isolated from the Pteridaceae, exhited marked mutagenicity in the modified Ames test with Salmonella typhimurian TA98 and A100 using a preincubation at pH 8.5 Illudins M and S, sesquiterpenes of the same illudane type from basidiomycetes, also exhibited mutagenicity. The structural requirements for mutagenicity are discussed.     

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes. I. Mutagenicity in Salmonella typhimurium

Ptaquiloside, a potent carcinogen of an illudane-type sesquiterpene glycoside isolated from Pteridium aquilium, and its related compounds, hypolosides having the same nucleus isolated from the Pteridaceae, exhited marked mutagenicity in the modified Ames test with Salmonella typhimurian TA98 and A100 using a preincubation at pH 8.5 Illudins M and S, sesquiterpenes of the same illudane type from basidiomycetes, also exhibited mutagenicity. The structural requirements for mutagenicity are discussed.     

Antigenic divergence in a primary rat hepatocyte culture after exposure to the hepatic carcinogen N-diethylnitrosamine

Using the indirect immunofluorescence method, the appearance of membrane hetero-organic antigens of kidney origin associated with the Zajdela hepatoma cells was observed on the surface of cells of the rat liver primary culture following a single hepatocarcinogen N-diethylnitrosamine (DENA) treatment before and after explantation. A correlation of antigenic alterations after a single DENA treatment in vivo and in vitro was discovered. No antigens under investigation were discovered in cultured hepatocytes of intact adult rats.     

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.     

Structure-activity relationships in the rat hepatocyte DNA-repair test for 300 chemicals

312 chemicals/mixtures were tested for genotoxicity in the rat hepatocyte/DNA-repair test. A variety of structure-activity relationships was evident. Of the 309 pure chemicals, 142 were positive. Of these, 43 were judged by IARC to have sufficient or limited evidence of carcinogenicity and none of the remainder was a proven noncarcinogen. Among the 167 negative chemicals, 44 were carcinogens. Some of these are known to be genotoxic in other systems, but based on several lines of evidence, many are considered to be epigenetic carcinogens that lack the ability to react with DNA and rather lead to neoplasia by nongenotoxic mechanisms.     

Comparison of 7 azo dyes and their azo reduction products in the rat and hamster hepatocyte primary culture/DNA-repair assays

Pursuant to the characterization of species differences in the effects of chemical carcinogens, several studies have demonstrated that hamster hepatocytes are more effective than rat hepatocytes in mediating the metabolic activation of certain chemicals to their genotoxic (i.e., mutagenic) derivatives. In the present investigation, a comparison of the amount of DNA repair induced in rat and hamster hepatocytes by 7 azo dyes and 7 aromatic amine azo reduction products of the dyes was performed using the primary hepatocyte culture/DNA repair (HPC/DR) assay. Congo Red and its azo reduction product, benzidine, were more potent inducers of DNA repair in hamster than in rat hepatocytes, whereas Trypan Blue and its reduction product, o-tolidine, were equipotent in the 2 hepatocyte systems. Evans Blue, another o-tolidine-based dye, elicited a greater DNA-repair response in hamster hepatocytes. The absolute potency of these dyes, however, was much less than their reduction products. o-Aminoazotoluene was the most potent of the dyes tested, and its DNA repair-inducing activity was much greater than that of its azo reduction products, o-toluidine and 2,5-diaminotoluene. Ponceau SX, which is carcinogenic in hamsters, but not in rats, was inactive in both hepatocyte systems. Dimethylaminobenzeneazo-1-naphthalene and its 2-naphthalene congener, as well as the 1- and 2-naphthylamine azo reduction products of these dyes, were more potent in hamster than in rat hepatocytes. However, the DNA repair-inducing activities of the parent dyes could not be entirely accounted for by the potencies of their respective naphthylamine derivatives. Taken together, these findings extend previous observations of the superior metabolic activation capabilities of hamster, relative to rat hepatocytes, and further demonstrate the utility of testing chemicals in both the hamster and rat HPC/DR assays.     

Genotoxic effects of a variety of sterigmatocystin-related compounds in the hepatocyte/DNA-repair test and the Salmonella microsome assay

The genotoxicity and mutagenicity of 11 fungal metabolites structurally related to sterigmatocystin were examined in the hepatocyte primary culture/DNA-repair test and the Salmonella microsome assay. 10 out of the mycotoxins, i.e. dihydrosterigmatocystin, 5-methoxysterigmatocystin, 5-methoxydihydrosterigmatocystin, 5,6-dimethoxysterigmatocystin, 5,6-dimethoxydihydrosterigmatocystin, sterigmatin, O-methylsterigmatocystin and O-acetylsterigmatocystin showed a positive response for DNA repair, suggesting their carcinogenic potency. 5-Methoxysterigmatocystin, 5,6-dimethoxysterigmatocystin demethylsterigmatocystin and O-acetylsterigmatocystin were mutagenic in TA100 of the bacterial mutagenicity assay with liver S9.     

The hepatocyte primary culture/DNA repair test using hepatocytes from several species

The hepatocyte primary culture/ DNA repair test, originally validated with rat hepatocytes, has been extended to use hepatocytes from other species including mouse, hamster, guinea pig, rabbit, monkey and human. Both qualitative and quantitative differences have been observed when chemicals are examined in the hepatocyte primary culture/DNA repair test using hepatocytes from more than one species. Examples are discussed that illustrate that the genotoxicity of a chemical can be a species-specific response and that multi-species testing permits a more complete assessment of genotoxicity.Abbreviations HPC hepatocyte primary culture - MOCA 4,4-methylene-bis-2-chloroaniline     

Evaluation of morpholine, 3-morpholinone, and N-substituted morpholines in the rat hepatocyte primary culture/DNA repair test

Although mature mammalian sperm are incapable of DNA repair, repair of damaged sperm DNA can occur after fertilization, as the sperm head decondenses and forms the male pronucleus. To quantify the cytogenetic effects of damage to sperm DNA we adapted the sister-chromatid exchange (SCE) test for use in early mouse embryos. After ultraviolet (UV) irradiation of sperm, eggs were fertilized in vitro and cultured for 2 cell cycles in medium containing fluorodeoxyuridine and bromodeoxyuridine; chromosomes were then prepared for SCE analysis. We found that UV-induced SCEs could be detected at the second cleavage division, and that eggs of different strains showed different frequencies of SCEs when fertilized by damaged sperm of a single strain. These results may indicate strain-specific differences in DNA repair of UV-induced DNA lesions by the early mouse embryo.     

Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens

The hepatocyte primary culture/DNA repair test was evaluated for its reliability using a series of coded samples. Among the 30 chemicals tested, 15 were general reference compounds and 15 were chemicals that had been tested for carcinogenicity in the U.S. National Cancer Institute Bioassay Program. The latter group were from the same lot that had been used for the in vivo testing and had also been tested for mutagenicity in the Ames test. From the group of 15 reference compounds, 5 were positive for DNA repair and all 5 were carcinogens. Of the 10 samples scored as negative, 4 were noncarcinogens and 6 were carcinogens. Among the 6 carcinogens were 3 compounds whose carcinogenicity probably does not involve the production of DNA damage. From the 15 coded chemicals that were tested for carcinogenicity by the NCI in long-term animal studies, 7 were scored as positive. 5 of these were judged carcinogenic in the in vivo bioassays and the other 2, which were also mutagenic in Salmonella, showed some indication of carcinogenicity. Of the 8 compounds that were scored as negative, 5 were noncarcinogenic. Among the 3 carcinogens that were not detected, there was at least one whose carcinogenicity probably does not involve DNA damage. Thus, the results of this study indicate that positive results in the hepatocyte primary culture/DNA repair test are highly specific for carcinogens and that the test is also highly sensitive in the detection of DNA-damaging genotoxic carcinogens.     

An assessment of the in vivo rat hepatocyte DNA-repair assay

The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described by Mirsalis et al, and its in vitro counterpart described earlier by Williams have been employed by us for 4 years. Our experience is that the in vivo assay performs as described in the literature. We have therefore concentrated in this initial paper on the key practical factors we have found to govern the assay sensitivity and reproducibility. This has been achieved by a discussion of the assay performance with two potent rat hepatocarcinogens [the novel azo compound 6-dimethylaminophenylazobenzthiazole (6BT) and the reference agent 2-acetylaminofluorene (2AAF)] and a non-carcinogen of similar structure to 6BT [5-dimethylaminophenylazoindazole (51)]. Assay responses were compared with the effect of these chemicals in the Salmonella mutation assay. We conclude that the in vivo liver UDS assay has a critical role to play as a complement to rodent bone marrow cytogenic assays when conducting assessment studies on agents defined as genotoxic in vitro. However, the in vivo assay is resource-consuming and false results could consequently arise due to incomplete evaluations. Methods to counteract this danger are discussed and criteria for assessing weak UDS responses are suggested.     

Genotoxicity of benzo[a]pyrene, 2-nitrofluorene and airborne particulates in the DNA-repair host-mediated assay

The genotoxic activity of benzo[a]pyrene (BAP), 2-nitrofluorene (NF) and airborne particulate matter was evaluated in the DNA-repair host-mediated assay after intraperitoneal or intratracheal administration. Dimethylnitrosamine (DMNA), used as a positive control, showed a genotoxic effect after both intraperitoneal and intratracheal administration, the strongest effect being found in liver, followed by lungs and kidneys, whereas a weak effect was observed in the spleen. In general no difference in genotoxicity was found between the 2 administration routes used. For BAP, although clearly positive in vitro, a moderate dose-dependent effect was found only in the liver after intraperitoneal administration. NF, which was positive in vitro both with and without a metabolizing system, produced no genotoxic effect in any of the organs tested after intraperitoneal administration. Extracts of airborne particulate matter which were genotoxic in vitro failed to cause a genotoxic effect in vivo by either route of administration. Possible explanations for the differences between the data obtained in vitro and in vivo are discussed.     

Genotoxicity in the hepatocyte/DNA repair test and toxicity to liver mitochondria of 1-hydroxyanthraquinone and several dihydroxyanthraquinones

1-Hydroxyanthraquinone and dihydroxyanthraquinones (alizarin, quinizarin, anthrarufin and chrysazin) were examined for genotoxicity in the hepatocyte/DNA repair test and for effects on oxidative phosphorylation in isolated rat liver mitochondria. Of the anthraquinone compounds tested, 1-hydroxyanthraquinone and 1,8-dihydroxyanthraquinone (chrysazin) induced DNA repair synthesis in rat hepatocytes, indicating their genotoxic activity. Only 1,2-dihydroxyanthraquinone (alizarin) exerted an uncoupling and inhibitory effect on mitochondrial respiration. The possible relationships of the genotoxic potencies and the uncoupling activities of these anthraquinones to their chemical structures are discussed.Abbreviations ADP adenosine-5-diphosphate - ETP electron transport particles - RC respiratory control - TdR thymidine deoxyribonucleotide - UDS unscheduled DNA synthesis     

Characterization of a primary hepatocyte culture system for toxicological studies

Summary An hepatocyte culture system was developed for potential use in toxicological studiesin vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox™ dishes in a modified Chee’s medium containing 1μM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system forin vitro toxicological studies. A patent has been filed for the use of the system described herein as anin vitro test kit.     

Lipoxygenase inhibitors induce rat hepatocyte apoptosis in primary culture

Arachidonic acid metabolites have been shown to have a wide range of effects on cell proliferation and viability. In this study, the effects of lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and caffeic acid (CA) on the viability of cultured rat hepatocytes (HC) were investigated. As a result, treatment with NDGA and CA for 4 h and 24 h decreased ALT release from HC and increased a number of apoptotic cells. Apoptosis inducing effects of general LO inhibitor NDGA were more pronounced, than those of 5-LO inhibitor CA. The results suggest that lipoxygenase pathway of arachidonic acid metabolism, in particular 5-LO, is essential regulator of hepatocyte survival and apoptosis.     

Calcium compartmentation and exchange rates in primary hepatocyte culture

We utilized a technique, previously used to study myocardial cells (G. A. Langer, J. S. Frank, and L. M. Nudd, 1979, Amer. J. Physiol. 237, H239-H246), to study 45Ca2+ isotope exchange kinetics in hepatocyte monolayers, cultured on scintillation disks, and perfused in a flow-through chamber. Isolated rat hepatocytes were plated directly on Primaria-coated disks impregnated with scintillation fluors which made up the walls of the perfusion chamber. Following the labeling of the cells with radioactive calcium (45Ca2+), to apparent asymptote, the washout of 45Ca2+ from the cells was measured. A large very fast turnover compartment, as well as small fast and slow turnover compartments, were identified in each experiment. Surface calcium (Ca2+) was determined by its displacement with 1 mM La3+ after asymptote had been reached during 45Ca2+ labeling (1.59 mmol Ca2+/kg dry wt). The rate constant for this compartment was faster than the washout of the chamber (greater than 3.4 min-1 with a t1/2 less than 12 s). The rate constants for the fast and slow exchangeable compartments were 0.11 min-1 (t1/2 = 6.5 min) and 0.013 min-1 (t1/2 = 56 min), respectively. The fast compartment contained 0.40 mmol Ca2+/kg dry wt and the slow compartment contained 0.27 mmol Ca2+/kg dry wt. Neither the fast nor the slow compartment was lanthanum displaceable. Release of 45Ca2+ in response to 100 microM phenylephrine, 10 nM angiotensin II, and 100-microM 2,5-ditert-butyl hydroquinone was measured during the washout phase. Ca2+ released by these compounds was determined to be 0.50 mmol 0.44, and 0.43 mmol Ca2+/kg dry cell wt, respectively. These agents had an effect only during the washout of the fast compartment. In conclusion, this novel technique of on-line measurement of 45Ca2+ exchange in hepatocyte monolayers identified three exchangeable compartments: (1) a very rapidly exchangeable surface compartment, (2) a fast "microsomal" hormone-releasable compartment, and (3) a slow, non-hormone-releasable compartment.