Mutagenicity Screening Research Articles

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23–63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1.

Mutagenic screening of some commonly used medicinal plants in Nigeria

The uses of medicinal plants have always been part of human culture. The World Health Organization estimates that up to 80% of the world's population relies on traditional medicinal system for some aspect of primary health care. However, there are few reports on the toxicological properties of most medicinal plants especially, their mutagenicity and carcinogenicity. Therefore, this research is to determine the mutagenic potentials of Morinda lucida [Oruwo (Root)], Azadirachta indica [Dongoyaro (Leaf)], Terapluera tetraptera [Aridan (Fruit)], Plumbago zeylanica [Inabiri (Root)], Xylopia aethiopica [Erunje (Fruit)], Newbouldia laevis [Akoko (Leaf)], Alstonia boonei [Ahun (Bark)], Enantia chlorantha [Awopa (Bark)], and Rauvolfia vomitoria [Asofeyeje (Root)] using the Allium cepa Linn. model and the modified Ames assay. Allium cepa model was used to determine the mean root length, mitotic index and chromosomal aberrations effects of these plants on onion bulbs using 0.1, 1, 5 and 10 mg/ml concentration of the plant extracts. The modified Ames test which is a modification of the standard Ames test as described by Ames et al. [Ames, B.N., McCann, J., Yamasaki, E., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Research 31, 347–364] was done using Escherichia coli (0157:H7) that has the phenotypic characteristics of glucose and lactose fermentation, motile, urease negative, indole positive and citrate negative. The results obtained from Allium cepa assay showed increasing root growth inhibition with increased concentration, decreasing mitotic index with increased concentration and chromosomal aberrations. The modified Ames test showed an alteration in the biochemical characteristics of Escherichia coli (0157:H7) for all plants except Rauvolfia vomitoria and Plumbago zeylanica. Three of the medicinal plants altered at least three of the normal biochemical characteristics thus demonstrating mutagenic potentials. The results of internationally accepted Allium cepa were comparable with the modified Ames test. However, a long term in vivo and dose dependent study should be carried out to validate these results and the findings should be communicated to drug and food regulatory body and also to the general public.

Inter-laboratory evaluation of the bioluminescent Salmonella reverse mutation assay using 10 model chemicals

We have developed the bioluminescent Salmonella reverse mutation assay as a tool for detecting mutagenicity applicable for high-throughput screening of new chemicals. In this study, we report the inter-laboratory evaluation of the assay using 10 model chemicals in five independent laboratories located in the USA (Groton, CT; Cambridge, MA and La Jolla, CA), Europe (Sandwich, Kent, UK) and Asia (Nagoya, Japan). The studies were performed in blinded fashion in all sites except for Groton and Cambridge laboratories. The chemicals were tested in at least three independent experiments using strains TA98-lux and TA100-lux in the presence and absence of metabolic activation. The results were statistically evaluated and compared to published results. Seven of the 10 compounds were positive in either TA98-lux and/or TA100-lux in the presence or absence of metabolic activation. The positive compound set included: nitrofurazone, 3-3'-dimethoxybenzidine, benzo[a]pyrene, 1,4-benzoquinone dioxime, 2-amino-5-nitrophenol, 2-bromo-4,6-dinitroaniline and busulfan. The remaining three compounds, namely, anthracene, crystal violet and benzyl chloride were negative in both Salmonella strains. Final results for individual compounds yielded 100% agreement among the laboratories and published data. Detailed comparison of all 40 individual test conditions yielded 93% (37 of 40) agreement among participating laboratories. We conclude that the bioluminescent Salmonella reverse mutation assay is a robust, accurate and economical higher throughput assay applicable for the mutagenicity screening of chemicals.

Flavouring Group Evaluation 15, Revision 1 (FGE.15Rev1) - Aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives from chemical group 22 - Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC).

Flavouring Group Evaluation 15, Revision 1 (FGE.15Rev1) - Aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives from chemical group 22 - Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC).

Biochemical and Genetic Analyses Provide Insight Into the Structural and Mechanistic Properties of Actin Filament Disassembly by the Aip1p–Cofilin Complex in Saccharomyces cerevisiae

Explication of the Aip1p/cofilin/actin filament complex may lead to a more detailed understanding of the mechanisms by which Aip1p and cofilin collaborate to rapidly disassemble filaments. We further characterized the actin-Aip1p interface through a random mutagenic screen of ACT1, identifying a novel Aip1p interaction site on actin. This finding is consistent with our current ternary complex model and offers insights into how Aip1p may disturb intersubunit contacts within an actin filament. In addition, site-directed mutagenesis aimed at interfering with salt bridge interactions at the predicted Aip1p-cofilin interface revealed hyperactive alleles of cof1 and aip1 that support the ternary complex model and suggest that conformational changes in cofilin structure may be transmitted to actin filaments, causing increased destabilization. Furthermore, these data support an active role for Aip1p in promoting actin filament turnover.

The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene-the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease - a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases - which in turn, are rarely incorporated into restriction/modification systems.

Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part I: Early Screening for Potential Human Mutagens

Results of genetic toxicology tests are used by FDA's Center for Drug Evaluation and Research as a surrogate for carcinogenicity data during the drug development process. Mammalian in vitro assays have a high frequency of positive results which can impede or derail the drug development process. To reduce the risk of such delays, most pharmaceutical companies conduct early non-GLP (good laboratory practices) studies to eliminate drug candidate with mutagenic or clastogenic activity. Early screens include in silico structure activity assessments and various iterations of the ultimate regulatory mandated GLP studies.

Evaluation of new 2,2′-dimethyl-5,5′-dipropoxybenzidine- and 3,3′-dipropoxybenzidine-based direct dye analogs for mutagenic activity by use of the Salmonella/mammalian mutagenicity assay

As part of a continuing study aimed at establishing structure–activity relationships and heuristic principles useful for the design of non-genotoxic azo dyes, a series of new direct dyes based on two non-mutagenic benzidine analogs, 2,2′-dimethyl-5,5′-dipropoxybenzidine and 3,3′-dipropoxybenzidine, were evaluated for mutagenic activity in Salmonella typhimurium strains TA98 and TA100. These strains are widely used for mutagenicity screening and have been shown to detect the mutagenic activity of benzidine analogs. While some toxicity was seen with some dyes at high doses, all of the dyes examined were judged non-mutagenic with and without metabolic activation in the standard Salmonella plate-incorporation assay. The results in the standard test are consistent with the properties of the diamines themselves. However, only one of the dyes was non-mutagenic when a reductive-metabolism pre-incubation assay was used. The results of this study suggest that although benzidine analogs are potential replacements for benzidine, there is a need to understand which mutagenic products are produced when reductive metabolism is present. There is also a need to know whether or not metal complexes of these dyes are mutagenic. Such information will allow the development of new non-mutagenic azo dyes.

In silico screening of chemicals for bacterial mutagenicity using electrotopological E-state indices and MDL QSAR software

Quantitative structure–activity relationship (QSAR) software offers a rapid, cost effective means of prioritizing the mutagenic potential of chemicals. MDL QSAR models were developed using atom-type E-state indices and non-parametric discriminant analysis. Models were developed for Salmonella typhimurium gene mutation, combining results from strains TA97, TA98, TA100, TA1535, TA1536, TA1537, and TA1538 ( n = 3228), and Escherichia coli gene mutation tests WP2, WP100, and polA ( n = 472). Composite microbial mutation models ( n = 3338) were developed combining all Salmonella, E. coli, and the Bacillus subtilis rec spot test study results. The datasets contained 74% non-pharmaceuticals and 26% pharmaceuticals. Salmonella and microbial mutagenesis external validation studies included a total of 1444 and 1485 compounds, respectively. The average specificity, sensitivity, positive predictivity, concordance, and coverage of Salmonella models was 76, 81, 73, 78, and 98%, respectively, with similar performance for the microbial mutagenesis models. MDL QSAR and discriminant analysis provides rapid and highly automated mutagenicity screening software with good specificity, sensitivity, and coverage that is simpler and requires less user intervention than other similar software. MDL QSAR modules for microbial mutagenicity can provide efficient and cost effective large scale screening of compounds for mutagenic potential for the chemical and pharmaceutical industry.

Electrochemical mutagen screening using microbial chip

Electrochemical microbial chip for mutagen screening were microfabricated and characterized by scanning electrochemical microscopy (SECM). Salmonella typhimurium TA1535 with a plasmid pSK1002 carrying a umuC'-'lacZ fusion gene was used for the whole cell mutagen sensor. The TA1535/pSK1002 cells were exposed to mutagen solutions containing 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2), mitomycin C (MMC) or 2-aminoanthracene (2-AA) and embedded in a microcavity (5nl) on a glass substrate using collagen gel. The beta-galactosidase expression on the microbial chip was electrochemically monitored using p-aminophenyl-beta-d-galactopyranoside (PAPG) as the enzymatic substrate. This system has several advantages compared with the conventional umu test: drastic reduction of the sample volume, less time-consuming for beta-galactosidase detection (free from substrate reaction time) and lower detection limit for the three mutagens (AF-2, MMC, 2-AA). Finally, a multi-sample assay was carried out using the microbial array chip with four microcavities.

Crystallographic Evidence for a New Ensemble of Ligand-Induced Allosteric Transitions in Hemoglobin: The T-to-THigh Quaternary Transitions,

A detailed description of hemoglobin cooperativity requires knowledge of the dimer-dimer interactions responsible for the low ligand affinity of the quaternary-T tetramer, the "quaternary-T constraints", along with stereochemical pathways that specify how ligand binding disrupts these quaternary constraints. The recent mutagenic screen of Noble et al. [Noble, R. W., et al. (2001) Biochemistry 40, 12357-12368] has identified the major region of quaternary constraint to be a cluster of residues at the alpha1beta2 interface that is centered at Trp37beta. In this paper, crystallographic studies are presented for most of the mutant hemoglobins studied by Noble et al. These crystallographic experiments identify structural transitions-referred to as T-to-T(High) transitions-between the quaternary-T structure of wild-type deoxyhemoglobin and an ensemble of related T-like quaternary structures that are induced by some mutations in the Trp37beta cluster and/or by exposing crystals of wild-type or mutant deoxyhemoglobins to oxygen. The T-to-T(High) quaternary transitions consist of a rotation of the alpha1beta1 dimer relative to the alpha2beta2 dimer as well as a coupled alphabeta dimer bending component that consists of a small rotation of the alpha1 subunit relative to the beta1 subunit (and a symmetry related rotation of the alpha2 subunit relative to the beta2 subunit). In addition, differences in subunit tertiary structure associated with the T-to-T(High) transitions suggest two stereochemical pathways (one associated with the alpha subunits and one associated with the betasubunits) by which ligand binding specifically disrupts quaternary constraints in the Trp37beta cluster. In the alpha subunits, ligand binding induces a shift of the heme iron producing tension in a chain of covalent bonds that extends from the Fe-N(epsilon)(2)His(F8)alpha1 bond to the peptide backbone bonds of residues His87(F8)alpha1 and Ala88(F9)alpha1. This tension induces an alpha-to-pi transition in the COOH-terminal end of the F-helix that shifts the beta-carbon of Ala88alpha1 by approximately 1.5 A directly into the side chain of Tyr140alpha1 (a key residue in the Trp37beta2 cluster). Collectively these structural changes constitute a relatively short pathway by which ligand binding forces Tyr140alpha1 into the alpha1beta2 interface disrupting quaternary constraints associated with the Trp37beta2 cluster. In the beta subunits, our analysis suggests a more extended energy transduction pathway in which ligand-induced beta1-heme movement triggers tertiary changes in the beta1 subunit that promote alpha1beta1 dimer bending that disrupts quaternary constraints in the Trp37beta2 cluster at the alpha1beta2 interface.

Identification and characterization of critical cis-acting sequences within the yeast Ty1 retrotransposon.

The yeast long terminal repeat (LTR) retrotransposon Ty1, like retroviruses, encodes a terminally redundant RNA, which is packaged into virus-like particles (VLPs) and is converted to a DNA copy by the process of reverse transcription. Mutations predicted to interfere with the priming events during reverse transcription and hence inhibit replication are known to dramatically decrease transposition of Ty1. However, additional cis-acting sequences responsible for Ty1 replication and RNA dimerization and packaging have remained elusive. Here we describe a modular mini-Ty1 element encoding the minimal sequence that can be retrotransposed by the Ty1 proteins, supplied in trans by a helper construct. Using a mutagenic screening strategy, we recovered transposition-deficient modular mini-Ty1-HIS3 elements with mutations in sequences required in cis for Ty1 replication and integration. Two distinct clusters of mutations mapped near the 5'-end of the Ty1 RNA. The clusters define a GAGGAGA sequence at the extreme 5'-end of the Ty1 transcript and a complementary downstream UCUCCUC sequence, 264 nt into the RNA. Disruption of the reverse complementarity of these two sequences decreased transposition and restoration of complementarity rescued transposition to wild-type levels. Ty1 cDNA was reduced in cells expressing RNAs with mutations in either of these short sequences, despite nearly normal levels of Ty1 RNA and VLPs. Our results suggest that the intramolecular interaction between the 5'-GAGGAGA and UCUCCUC sequences stabilizes an RNA structure required for efficient initiation of reverse transcription.

Alternatives to Toxicity Testing in Animals: Challenges and Opportunities

We have learned over time that the development of successful alternative methods in toxicology testing requires the successful integration of three elements: First, there must be a solid foundation of understanding the basic biology and toxicology of the tissues and organs being studied. Second, in vitro platforms must be available that can be modified to make them amenable for toxicity testing. Third, one needs to convince the scientific community, which is skeptical by nature and training (and rightfully so), that the alternative methods fulfill their intended purpose and have been rigorously validated. In vitro mutagenicity screening methods have been used for many years and are a good illustration of these three points. Initially, the basic biology that needed to be understood was that DNA is the molecular basis for heredity and that mutations are, in fact, manifestations of damage to the DNA. Furthermore, several types of mutations (e.g., point mutations, insertions, deletions) require the development of different in vitro models. In vitro platforms, the second element, were adapted from extensive research into the molecular biology of prokaryotes, and later, eukaryotic cells. The third element involved years of assay standardization, replication of results in multiple laboratories, and comparisons with in vivo results. For more complex end points, the development of alternatives has been a daunting task. Even supposedly simple targets for replacement, such as the Draize test for eye irritation, have proved difficult to model in vitro and progress through successful external validation despite major efforts by the European Centre for the Validation of Alternatives (ECVAM), industry trade associations, individual companies, and academia. An extensive list of in vitro models that have been proposed as alternatives to the Draize test has been published E s s a y o n Toxicity Testing in Animals

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex.

Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1). We have determined a high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds. A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains.

Identification of a zebrafish model of muscular dystrophy.

1. Large-scale mutagenic screens of the zebrafish genome have identified a number of different classes of mutations that disrupt skeletal muscle formation. Of particular interest and relevance to human health is a class of recessive lethal mutations in which muscle differentiation occurs normally, but is followed by tissue-specific degeneration reminiscent of human muscular dystrophies. 2. We have shown that one member of this class of mutations, sapje (sap), results from mutations within the zebrafish orthologue of the human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology by disrupting the link between the actin cytoskeleton and the extracellular matrix in skeletal muscle cells. 3. We have found that the progressive muscle degeneration phenotype of sapje-mutant zebrafish embryos is caused by the failure of somitic muscle attachments at the embryonic myotendinous junction (MTJ). 4. Although a role for dystrophin at the MTJ has been postulated previously and MTJ structural abnormalities have been identified in the dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure is thus far lacking. Therefore, the sapjre mutation may provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases. In the present review, we discuss this finding in light of previously postulated models of dystrophin function.

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incanaR. Br. (Brassicaceae)

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.

Mutation of the zebrafish choroideremia gene encoding Rab escort protein 1 devastates hair cells.

To identify genes important for hair-cell function, we conducted a mutagenic screen in zebrafish. Larvae from one mutant line, ru848, were unresponsive to acoustic stimuli and unable to balance. The mutation results in a 90% reduction in hair-cell number and partial retinal degeneration by 5 days postfertilization. We localized the recessive ru848 mutation by positional cloning to the zebrafish homolog of the human Choroideremia gene, which encodes Rab escort protein 1. This protein is essential for the normal prenylation of Rabs. Mutations in the human gene induce choroideremia, a disease marked by slow-onset degeneration of rod photoreceptors and retinal pigment epithelial cells. The degenerative phenotype resulting from a null mutation in the zebrafish gene indicates that hair cells and retinal cells require Rab escort protein 1 for survival.

The novel mouse microphthalmia mutations Mitfmi-enu5 and Mitfmi-bcc2 produce dominant negative Mitf proteins

Mutations in the microphthalmia-associated transcription factor ( Mitf) gene affect the development of different cell types, including melanocytes, osteoclasts, and retinal pigmented epithelial cells of the eye. Many different mutations at the locus are known and since they affect the phenotype to different extents they form an allelic series. The Mitf protein is a member of the Mitf-Tfe subfamily of basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factors and binds the 6-bp canonical CAC/TGTG sequence (E box) as either a homodimer or a heterodimer with related proteins. The many Mitf mutations have provided important insights into the in vivo behavior of a bHLH-Zip protein. Here we describe the phenotype of two new semidominant Mitf mutations recovered in recent mutagenic screens, Mitf mi-enu5 and Mitf mi-bcc2 ; determine the molecular lesions involved; and show that the mutant proteins act in a dominant negative fashion in vitro. The novel mutations are phenotypically distinct from previously known Mitf mutations.

The development of an in vitro screening strategy for topically applied products

The objective of this study was the pharmaco-toxicological understanding of the constituents of an authenticated herbal mixture. The mixture was prepared by maceration in ethanol and subsequent dilution to produce a topically applied lotion, for which the intended target conditions are psoriasis and eczema. A three-tiered in vitro screening strategy was adopted for evaluating this product, comprising cytotoxicity assays; mutagenicity screening and therapeutic evaluation. Viability assays performed with dilutions of both the herbal concentrate and final product on organotypic cell lines indicated that neither preparation acted as an irritant. Genotoxicity screening using six strains of Salmonella typhimurium showed no mutagenic potential, and furthermore significant anti-microbial activity was evident. Therapeutic evaluation involved assessing the antioxidant potential of the extract, which can be correlated to an anti-inflammatory effect. Nitroblue-tetrazolium (NBT) assay results indicate that the extract can reduce superoxide anion generation by 45%. The extract also increased cell viability on exposure to hydrogen peroxide by 28%, illustrating its dismutation potential. A 3-D skin culture system, EpiDerm™, released 3000 μg/ml upon exposure to the extract, implying that the components enhance arachidonic acid metabolism. Overall, it may be concluded that the herbal extract is sufficiently non-toxic for dermal application and possesses anti-inflammatory activity.

Hepatocyte Culture as an In Vitro Model for Evaluating the Hepatotoxicity of Food-Borne Toxicants and Microbial Pathogens: A Review

Hepatocyte culture is a well-established, well-characterized, and widely used in vitro tool for pharamacological and toxicological studies. The hepatocytes of a wide range of species, including humans, can be maintained in culture with a high metabolizing capacity for several days under closely controlled and easily manipulated conditions. Numerous studies have reported good correlation between in vitro hepatocytes and in vivo siutations. They have been widely used for studies of the metabolism and of the toxicity and mechanisms of action of chemicals and drugs, for the screening of mutagens, carcinogens, and micotoxins, for virulence assessment of microbial pathogens and viruses, for qualitative and quantitative interspecies comparison, and for genomics and proteiomics studies. Hepatocytes, especially human hepatocytes, are used for preclinical drug evaluation and screening as well as for studies of drug metabolism, toxicity, interactions, and structure-activity relationships. They can be used for evaluating the hepatotoxicity of herbal products, dietary supplements, food additives, food-borne toxicants, and microbial pathogens. The results obtained from such in vitro screenings can be used for in vivo studies to asses the safety of test materials of interest. At the present time, there is insufficient evidence for their use in quantitative risk assessment. However, they are suitable for use in qualitative hazard assessment, which may be used for quantitative risk analysis.