Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; current state of knowledge.

This article gives an overview of the results of genotoxicity tests, which have been conducted within the last 5 years with the human liver cell line HepG2. It is an update of an earlier review from 1998 (by Knasmüller et al.). In addition, a number of publications are discussed which are relevant for the use of human derived liver cell lines in genetic toxicology. They concern the establishment of new endpoints, the development of new cell lines and possible pitfalls and problems. HepG2 cells have been used to test a wide variety of compounds over the last years. The most interesting observations are that the cells are highly sensitive toward polycyclic aromatic hydrocarbons and that genotoxic effects are seen with a number of carcinogenic mycotoxins, that give negative results in other in vitro assays. Carcinogenic metals such as As and Cd caused positive results as well, whereas only marginal or negative results were seen with nitrosamines. The low sensitivity toward these latter carcinogens is probably due to a lack of cytochrome P4502E1 which catalyses their activation. Also, a number of structurally different synthetic pesticides as well as bioactive plant constituents ("natural pesticides") have been tested and with some of them genotoxic effects were found. In most experiments, the formation of micronuclei was used as an endpoint; however also the single cell gel electrophoresis assay is increasingly used. Several transfectant lines of HepG2 have been constructed which express increased levels of phase I enzymes (such as CYP1A1, CYP1A2, CYP2E1 etc.); furthermore, cell lines became available which express human glutathione-S-transferases. These new clones might be particularly useful for the investigation of specific classes of genotoxicants and also for mechanistic studies. Apart from HepG2 cells, a number of other human derived liver cell lines have been isolated, but so far no data from genotoxicity experiments are available, except for Hep3B cells, which were compared with HepG2 and found to be less sensitive in general. Studies with HepG2 clones of a different origin indicate that the cells differ in regard to their sensitivity toward genotoxicants; also medium effects and the cultivation time might affect the outcome of genotoxicity studies. Overall, the results support the assumption that HepG2 cells are a suitable tool for genotoxicity testing.

The genotoxicity of unsubstituted and nitrated polycyclic aromatic hydrocarbons.

To determine the DNA damaging properties of unsubstituted and substituted polycyclic hydrocarbons, 61 aromatic and heterocyclic compounds were examined for the induction of the SOS system in E. coli PQ37. PAH such as benzo[ghi]fluoranthene, benzo[j]fluoranthene, benzo[c]phenanthrene, benzo[a]pyrene, chrysene, dibenzo[a,1]pyrene, fluoranthene and triphenylene showed relatively high genotoxicity. With respect to the nitroarenes, the highest genotoxic potencies were exhibited by the dinitropyrenes. The SOS-inducing potency of nitroarenes increased from the bicyclic to the tetracyclic ring system. Additionally, it was seen that any increase in the extent of nitration is paralleled by an increase of genotoxicity. Whereas PAH required metabolic activation by hepatic cytochrome P-450 enzymes, nPAH were direct-acting genotoxicants.

Genotoxicity of polycyclic musk fragrances in the sister-chromatid exchange test.

The synthetic polycyclic musk fragrance compounds Galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclo-penta-(g)-2-++ +benzopyrane, Tonalide (7-acetyl-1,1,3,4,4,6-hexamerthyltetraline), Celestolide (4-acetyl-1,1-dimethyl-6-tert, butylindane), Phantolide (6-acetyl-1,1,2,3,3,5-hexamethylindane), Cashmeran (6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H) indanone) and Traseolide (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane) are widely used as fragrance ingredients in perfumes, lotions and detergents; as food additives in cigarettes and fish baits. Several studies identified polycyclic musk fragrances in aquatic environment samples, human milk and human adipose tissue as highly lipophil with human lymphocytes.

Micronucleus formation in human lymphocytes and in the metabolically competent human hepatoma cell line Hep-G2: results with 15 naturally occurring substances.

To examine the concordance of two metabolizing systems for use in genotoxocity testing with the micronucleus test, 15 naturally occurring substances (arecoline, the plant extract aristolochic acid, beta-asarone, benzyl acetate, coumarin, emodine, isatidine dihydrate, monocrotaline, psoralen, reserpine, retrorsine, safrole, sanguinarine chloride, tannin and thiourea) were tested for their genotoxicity in the cytokinesis-block micronucleus test in vitro with human lymphocytes and in the presence and the absence of an exogenous metabolizing system from rat liver S9-mix and the metabolically competent human hepatoma cell line Hep-G2. Arecoline, the plant extract aristolochic acid, psoralen and tannin caused a significant increase in the number of micronuclei in human lymphocytes in the presence and the absence of an exogenous metabolising system from rat liver S9-mix and the metabolically competent human hepatoma cell line Hep-G2. A significant increase in the number of micronuclei with beta-asarone, coumarin, monocrotaline and retrorsine could be detected in the presence of S9-mix and the cell line Hep-G2. Benzyl acetate, emodine, isatidine dihydrate, reserpine, safrole, sanguinarine chloride and thiourea did not reveal any micronucleus inducing activity in either human lymphocytes or in Hep-G2. In addition to the other Hep-G2 results in the literature, this human hepatoma cell line could have a useful potential in the in vitro micronucleus test.

Genotoxicity of nitro musks in the micronucleus test with human lymphocytes in vitro and the human hepatoma cell line Hep G2.

The nitro musk compounds musk xylene (1-tert.-butyl-3,5-dimethyl-2,4,6-trinitrobenzene), musk ketone (4-tert.-butyl-3,5-dinitro-2,6-dimethylacetophenone), musk ambrette (1-tert.-butyl-4-methyl-6-methoxy-3,5-dinitrobenzene), musk moskene (1,1,3,3,5-pentamethyl-4,6-dinitroindane) and musk tibetene (1-tert.-butyl-3,4,5-trimethyl-2,6-dinitrobenzene) were tested for their genotoxic activity in the micronucleus test (MN) with human lymphocytes in vitro and the human hepatoma cell line Hep G2. Compound concentrations were employed up to cytotoxic doses. Musk xylene, musk ketone, musk ambrette, musk moskene and musk tibetene revealed no genotoxicity in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2.

Testing of SOS induction of artificial polycyclic musk fragrances in E. coli PQ37 (SOS chromotest).

Synthetic fragrances are widespread in the environment. Residues were found in animals, human tissues and breast milk. Therefore, six artificial polycyclic musk fragrances--Galaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and Traseolide--were tested for SOS induction using the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) in the presence (+S9) and absence (-S9) of an exogenous metabolizing system. All compounds tested exhibited no SOS inducing potency with the SOS chromotest. These results could be rated as one indicator of the biological inactivity of this group of compounds with respect to genotoxicity.

Genotoxicity of selected pesticides in the mouse bone-marrow micronucleus test and in the sister-chromatid exchange test with human lymphocytes in vitro.

Selected pesticides (aldicarb, 1,3-dichloropropene, methidathion, parathion, triadimefon, vinclozolin) were tested for their clastogenic and aneugenic activities in the mouse bone-marrow micronucleus (MN) test in vivo and for their sister-chromatid exchange-inducing activities in human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system from rat-liver S9. 1,3-Dichloropropene significantly increased the frequencies of micronucleated polychromatic erythrocytes (PCE) in bone-marrow cells of female mice from 3.3 MN/1000 PCE to 15.3 MN/1000 PCE (187 mg per kg body weight). 1,3-Dichloropropene (100 microM) induced 16.0 SCE/metaphase after 24 h of incubation as compared with the basal rate of 11.2 SCE/metaphase (-S9) and of 15.4 SCE/metaphase as compared with 10.5 SCE/metaphase of the control (+S9). These values were statistically significantly different from each other. The other pesticides tested did neither increase the rate of micronuclei significantly in polychromatic erythrocytes in male nor in female animals. Aldicarb and methidathion induced a significant increase in SCEs in human lymphocytes in vitro only without the metabolic activating system: aldicarb, 5 microM, 24 h incubation: 15.5 SCE/metaphase; control: 12.6 SCE/metaphase; methidathion, 100 microM, 24 h incubation: 15.8 SCE/metaphase, control: 11.1 SCE/metaphase. Parathion, triadimefon and vinclozolin did not have any SCE-inducing effects.

Sources of variability of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest).

To determine the variability in test results obtained with the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) when varying the test protocol, we examined the influences of sodium dodecylsulfate (SDS) concentrations, of buffer pH and composition on the enzyme assays, the effects of E. coli PQ37 density and culture conditions on the expression and/or determination of alkaline phosphatase (ap) and beta-galactosidase (beta-g) activities, the calculated induction factors (IF) and the SOS-inducing potentials (SOSIP). Initially, we used 0-190 ng (0-1 nmole) 4-nitroquinoline-1-oxide (4-NQO) as a reference compound for the standard procedure in the absence of metabolic activation. Subsequently, to evaluate the results of protocol variations we examined several mutagenic compounds of differing chemical classes using both the standard and a modified assay procedure. We observed the highest enzyme activities using 1 mg SDS per tube and calibrating the ap buffer to pH 8.05 and the beta-g buffer to pH 7.75. The longer the incubation period, the higher the enzyme activities. However, with respect to IF and SOSIP there is no reason to incubate in excess of 90 min. We found no significant differences in the IF and SOSIP values when varying substrate conversion times. There was, however, a definite decrease in beta-g activity when extended substrate incubation times were used. Higher enzyme activities are obtained when the bacterial count is increased. Using lower bacterial counts the enzyme activities decreased, but the sensitivity of E. coli towards genotoxic compounds increased.

Genotoxicity of polycyclic aromatic hydrocarbons in Escherichia coli PQ37.

In the present investigation, 32 polycyclic aromatic hydrocarbons (PAHs) were tested for genotoxicity in E. coli PQ37 using the standard tube assay of the SOS chromotest. PAHs such as benzo[ghi]fluoranthene, benzo[j]fluoranthene, benzo[a]pyrene, chrysene, dibenzo[a,l]pyrene, fluoranthene and triphenylene exhibited high genotoxicity when incubated in the presence of an exogenous metabolic activation mixture. The results were compared to those obtained with the Salmonella/microsome test.

In vitro genotoxicity of polycyclic musk fragrances in the micronucleus test.

The synthetic polycyclic musk fragrance compounds galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-b enzopyrane), tonalide (7-acetyl-1,1,3,4,4,6-hexamerthyltetraline), celestolide (4-acetyl-1,1-dimethyl-6-tert-butylindane), phantolide (6-acetyl-1,1,2,3,3,5-hexamethylindane), cashmeran (6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H) indanone) and traseolide (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane) were examined for their genotoxicity in the micronucleus test (MNT) with human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system containing rat liver S9 and the metabolically competent human hepatoma cell line Hep G2. Compound concentrations were employed up to cytotoxic doses. Galaxolide, tonalide, celestolide, phantolide, cashmeran and traseolide revealed no genotoxicity in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2.

Assessment of a possible genotoxic environmental risk in sheep bred on grounds with strongly elevated contents of mercury, arsenic and antimony.

A part of Northern Palatinate country (Germany) was formerly influenced by mercury mining. Today, in many cases agricultural and housing areas are placed onto or near to former dump grounds of rubble. In the soil of these areas the concentration of mercury, arsenic and antimony was found ranging from basic natural contents up to strongly elevated levels. In a biomonitoring project, sheep bred on grounds contaminated with mercury (range 1-435 mg Hg/kg dry matter), arsenic (range 17-147 mg As/kg dry matter) and antimony (range 2-15 mg Sb/kg dry matter) were taken as example on the uptake of these elements from the environment and for possible effects of this exposure. Significantly elevated mercury levels were found in wool of one collective of exposed sheep (0.107 mg/kg mean vs. 0.048 mg/kg mean, p < 0.001, U-test). Surprisingly, the arsenic content of wool taken from sheep bred in the urban referential area was approx. 10 times higher than that of the sheep bred on the grounds contaminated with arsenic (0.57 mg/kg mean vs. 0.051 mg/kg mean, p < 0.001, U-test). In general, element concentrations in the examined blood samples were low and the differences between the collectives were small: mercury was found in concentrations ranging from 0.9 microgram/l up to 2.0 micrograms/l (means), arsenic and antimony were generally found in concentrations below 1 microgram/l. Neither in the alkaline elution technique nor in the sister chromatid exchange (SCE) analysis significant increases in the rate of DNA-damaging effects between the different sheep collectives were detected. This indicates that the transfer rate of genotoxic compounds of mercury, arsenic or antimony from the environment is too low to register effects with AFE and SCE although the soil was highly contaminated.

SOS induction of selected naturally occurring substances in Escherichia coli (SOS chromotest).

Naturally occurring substances were tested for genotoxicity using a modified laboratory protocol of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) in the presence and in the absence of an exogenous metabolizing system from rat liver S9-mix. Aristolochic acid I, II, the plant extract aristolochic acid and psoralene were genotoxic; cycasine, emodine, monocrotaline and retrorsine were classified as marginal genotoxic in the SOS chromotest in the absence of S9-mix. In the presence of an exogenous metabolizing system from rat liver S9-mix aristolochic acid I, the plant extract, beta-asarone, cycasin, monocrotaline, psoralen and retrorsine showed genotoxic effects; aristolochic acid II marginal genotoxic effects. Arecoline, benzyl acetate, coumarin, isatidine dihydrate, reserpine, safrole, sanguinarine chloride, senecionine, senkirkine, tannin and thiourea revealed no genotoxicity in the SOS chromotest either in the presence or in the absence of an exogenous metabolizing system from rat liver S9-mix. For 17 of 20 compounds, the results obtained in the SOS chromotest could be compared to those obtained in the Ames test. It was found that 12 (70.6%) of these compounds give similar responses in both tests (6 positive and 6 negative responses). The present investigation and those reported earlier, the SOS chromotest, using E. coli PQ37, was able to detect correctly most of the Salmonella mutagens and non-mutagens.

Lack of Mutagenicity of Polycyclic Musk Fragrances in Salmonella typhimurium.

Synthetic musks are widely used as fragrances and therefore found in various environmental samples. Residues were identified in river and waste water, animal and human tissues and breast milk. In the present study, six artificial polycyclic musk fragrances-Galaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and Traseolide-were tested for mutagenicity using the Salmonella/mammalian-microsome assay with Salmonella typhimurium strains TA97, TA98, TA100 and TA102 in the presence (+S-9) and absence (-S-9) of an exogenous metabolizing system. All compounds tested exhibited no mutagenicity in the Salmonella assay. These results could be rated as one indicator of the biological inactivity of this group of chemicals with respect to genotoxicity.

Methods of in vitro toxicology.

In vitro methods are common and widely used for screening and ranking chemicals, and have also been taken into account sporadically for risk assessment purposes in the case of food additives. However, the range of food-associated compounds amenable to in vitro toxicology is considered much broader, comprising not only natural ingredients, including those from food preparation, but also compounds formed endogenously after exposure, permissible/authorised chemicals including additives, residues, supplements, chemicals from processing and packaging and contaminants. A major promise of in vitro systems is to obtain mechanism-derived information that is considered pivotal for adequate risk assessment. This paper critically reviews the entire process of risk assessment by in vitro toxicology, encompassing ongoing and future developments, with major emphasis on cytotoxicity, cellular responses, toxicokinetics, modelling, metabolism, cancer-related endpoints, developmental toxicity, prediction of allergenicity, and finally, development and application of biomarkers. It describes in depth the use of in vitro methods in strategies for characterising and predicting hazards to the human. Major weaknesses and strengths of these assay systems are addressed, together with some key issues concerning major research priorities to improve hazard identification and characterisation of food-associated chemicals.

Lead contamination in tap water of households with children in Lower Saxony, Germany.

Lead has numerous acute and chronic adverse effects on human beings. This is especially true for infants and children. The main path of lead ingestion in children can be different according to housing and living situation. The intake of lead through drinking water is commonly due to metal corrosion. The users plumbing can be an important factor. In recent years, many lead pipes in Germany have been replaced by pipes made of an alternative material. The aim of this study is to assess the present state of drinking water contamination and the resulting exposure of infants to lead. For this purpose mothers of new-born babies were offered a free examination of their drinking water. After a written declaration of consent had been obtained and after the infant in question had reached an age of 3 months, a stagnation sample of cold tap-water after overnight stagnation together with a random daytime sample was obtained from the family. The collected samples were analysed by atomic absorption spectrometry for their lead concentration. In total, 1485 samples from households were collected. Of the 1434 stagnation samples, 3.1% had lead concentrations greater than 0.01 mg/l (recommended limit of the WHO) and 0.6% had concentrations above the limit of the German drinking water regulation (0.04 mg/l). The values for the 1474 random daytime samples were 2.1% above 0.01 mg/l and 0.2% greater than 0.04 mg/l, respectively. By region, the areas Bovenden, Friedland, Duderstadt, Northeim and Rosdorf were particularly affected. The highest measured concentrations of lead in the stagnation samples were 0.11 mg/l and 0.15 mg/l in the random daytime samples, respectively.

Nitro musk compounds genotoxic activity : Genotoxicity testing of nitro musks with the SOS-chromotest and the sister-chromatid exchange test.

Five nitro musk compounds are widely used as fragrance ingredients in perfumes, lotions and detergents; as food additives in cigarettes and fish baits, and in such technical products as herbicide formulations and explosives. Several studies identified nitro musk compounds in aquatic environment samples, human milk and fat samples as highly lipophilic and persistent bioaccumulating environmental pollutants. To examine the compounds for genotoxic activity, musk xylene (1-tert.-butyl-3, 5-dimethyl-2, 4, 6-trinitrobenzene), musk ketone (4-tert.-butyl-3, 5-dinitro-2, 6-dimethylacetophenone), musk ambrette (l-tert.-butyl-4-methyl-6-methoxy-3, 5-dinitrobenzene), musk moskene (l, 1, 3, 3, 5-pemamethyl-4, 6-di-nitroindane) and musk tibetene (1-tert.-butyl-3, 4, 5-trimethyl-2, 6-dinitrobenzene) were tested for SOS inducing potency in the SOS chromotest with E. coli PQ37 and for sister-chromatid exchange inducing activities in human lymphocytes in vitro both in the presence and absence of an exogenous metabolizing system from rat liver S9-Mix. Nitro musks revealed no genotoxicity either in the SOS chromotest with E. coli PQ37 or in the sister-chromatid exchange test with human lymphocytes.

[Mutagenicity of mixtures of halogenated aliphatic hydrocarbons and polycyclic aromatic hydrocarbons in the Ames test with TA98 and TA100]. / Untersuchungen zur Mutagenität von Substanzgemischen aus leichtflüchtigen Organohalogenen und polyzyklischen Aromaten im Ames-Test mit TA98 und TA100.

Within the framework of the assessment of the genotoxic potential of environment samples the Salmonella-microsome-test (Ames-test) is often used as a screening-test. It is one of the most applied biotest systems and possesses a large scientific acceptance. Because most environment samples are mixtures of various substances, possible effects resulting from the combination should be taken into account with regard to the mutagenic potential. In this context we investigated eight polycyclic aromatic hydrocarbons each combined with six halogenated aliphatic hydrocarbons as to their mutagenicity in the Salmonella-microsome-test with TA98 and TA100. For an exogenous metabolizing system, Arochlor 1254 induced rat liver S9-mix was used. Benz-a-pyrene in combination with bromodichloromethane (Ames neg. in TA98 and TA100 +S9) showed an increase in the number of the revertants up to 25% in TA98 and TA100 (+S9). Carbon tetrachloride (Ames neg. in TA98 and TA100 +S9) showed in TA100 (+S9) an increase in the number of the revertants of 18% at most. In the combination 3-methylcholanthrene with dichloromethane the number of revertants in TA98 (+S9) increased by 25% and in TA100 (+S9) by 18%. Hexachloroethane (weakly mutagenic in TA98 +S9) in combination showed in TA98 (+S9) a slightly increased number of revertants with benz-a-pyrene as well with 3-methylcholanthrene. All the other substances tested (chrysene, phenanthrene, anthanthrene, dibenz-a, i-pyrene, triphenylene, fluoranthene) in combination with either tetrachloroethylene or trichloroethene did not cause an increase in mutagenicity.

In vivo genotoxicity of selected herbicides in the mouse bone-marrow micronucleus test.

The herbicides alachlor, atrazine, terbuthylazine, gluphosinate-ammonium, isoproturon, pendimethaline and trifluralin were tested for genotoxicity in the mouse bone-marrow micronucleus test (MNT). Both atrazine and trifluraline caused a significant increase in the number of micronuclei at doses of 1,400 mg/kg body weight in female mice only. Alachlor, terbuthylazine, gluphosinate-ammonium, isoproturon and pendimethaline did not have any genotoxic effect in the mouse bone-marrow micronucleus test in either female or male animals.

[Nitrite formation and nitrosation potential of heterotrophic biological denitrification of drinking water in laboratory conditions]. / Untersuchungen zur Nitritbildung und zum Nitrosierungspotential bei der heterotrophen biologischen Denitrifizierung von Trinkwasser unter Laborbedingungen.

In a laboratory construction for heterotrophic biological denitrification of drinking water treatment, the formation of nitrite, the potency of nitrosation and the genotoxic activity were tested. Parameter as nitrate concentration, the water flow rate in the system, nitrite and morpholine addition and the pH-value were checked. For testing the potency of nitrosation and formation of nitrite in the reactor we took morpholine, a fast nitrosing amine. The results show for the break down rate of nitrate, there is no influence of the initial nitrate concentration (80-195 mg/L), the nitrite addition (5-20 mg/L) and the water flow rate (45-100 min) in the system. pH-values below 5.5 showed a little break down rate of nitrate. There was no correlation between the starting point of nitrate concentration and the formation of nitrite, although there was a positive correlation between the length of stay and the formation of nitrite. Nitrite concentrations of 5 mg/L with morpholine concentrations of 5 and 10 mg/L didn't show detectable formation of nitrosomorpholine. The analyses of different watertests in the construction didn't show significant results for DNA damage by the sister-chromatid exchange (SCE). The results of the Salmonella microsome assay (tester strain TA1535) didn't show any mutagenic effects relating to the potency of nitrosation. According to our experiments the potency of generalising nitrosamides or nitrosamines by drinking water denitrification seems to be low. There is no final assessment of detriment to health by denitrifying drinking water.

Human effect monitoring in cases of occupational exposure to antineoplastic drugs: a method comparison.

OBJECTIVES: To investigate whether DNA damage increased in subjects possibly exposed to high amounts of antineoplastic agents. METHODS: The level of genetic damage was determined in peripheral mononuclear blood cells with the sister chromatid exchange test, the alkaline elution technique, and the cytokinesis block micronucleus test. RESULTS: The supposed increased exposure of the study subjects was caused by a malfunction of a safety hood resulting in leakage of air during preparation of an infusion of an antineoplastic drug. Two months after a new safety hood was installed, the frequencies of micronuclei and sister chromatid exchanges of exposed nurses (n = 10) were still significantly increased when compared with a matched control group (p < 0.01 and p < 0.05, one sided Wilcoxon test, respectively). In a second examination seven months later, the frequency of micronuclei had significantly decreased to control values (p < 0.05, one sided Wilcoxon test, n = 6). Moreover, the study subjects who smoked (n = 8) had significantly increased frequencies of micronuclei and sister chromatid exchanges (p < 0.01 and p < 0.05, one sided U test, respectively). No differences in the rate of DNA damage could be detected with the alkaline elution technique. CONCLUSIONS: Control measures on the level of biological effect should be performed regularly to ensure maximum safety precautions for workers potentially exposed to genotoxic agents.