The place of the host-mediated assay

Techniques are available for the detection of chromosomal mutations in mammals, but only the specific locus, coat colour test is presently available for attempting to detect directly gene mutations in mammals. A plethora of tests has been developed to make this detection possible, but always indirectly and usually with micro-organisms. In order to compensate for the metabolic deficiencies (compared with mammals) of micro-organisms, two approaches to the problem have been attempted: (a) tissue-mediated assays and (b) host-mediated assays.In host-mediated assays, cells (bacteria, yeast, Neurospora, mammalian cells) are injected into the peritoneal cavity, testes or blood of mice or rats, then, after a suitable incubation period, the cells are recovered from the peritoneal cavity, testes, or, when intravenous inoculation is used, from liver, lungs or kidneys. Recovered cells are assayed for survival and mutation frequency.High doses to the host mammal often have to be used in order to ensure adequate exposure of the indicator cells to the test agent. The test, then, suffers from insensitivity. Consequently, test results are biased towards false-negatives.Variability of the test results is another problem of the assay. On the other hand, there are certain applications where its value has been demonstrated. Examination of the effects of orally administered compounds may be the most important use of the technique.More generally valuable in hazard detection may be an extension of the original host-mediated assay, i.e., urine analysis for mutagenic activity. The metabolite concentrations are often high in urine, but the unstable and probably more important metabolites (from a toxicological view point) are poorly represented. Monitoring of human populations exposed to suspected mutagens is, however, possible with this technique.     

Mutagenicity testing with eukaryotic microorganisms

The different genetic end-points which can be tested to detect genotoxicity of chemicals in fungi and especially in the yeast Saccharomyces cerevisiae are described. They include reversion and forward mutation, mitochondrial deletions and point mutations, mitotic or meiotic intra- and intergenic recombination, chromosomal non-disjunction and aneuploidy. Several factors known to affect the response to genotoxic agents such as the growth parameters, the repair ability, the cells permeability, etc., are discussed. The recent validation studies on the mutagenic and recombinogenic activities of a number of chemicals indicate that within the battery of rapid, low cost and quantitatively reliable tests, the yeast system can be profitably used.     

Integration of Genotoxic and Population Genetic Endpoints in Biomonitoring and Risk Assessment

Genetic ecotoxicology is a multifaceted discipline that examines the effects of xenobiotic compounds on the structure and function of DNA. This paper discusses the role of genetic ecotoxicology in environmental biomonitoring and risk assessment. Genetic ecotoxicology may include somatic effects (e.g., DNA damage) or population genetic effects (changes in genetic diversity or gene frequencies). Traditionally, genetic ecotoxicology studies have focused on either one of these sub-disciplines, but integration of these two approaches would be advantageous for three reasons. First, at the population level, concordant responses between changes in population genetic structure and elevated levels of DNA damage may provide evidence that the population genetic changes are influenced by exposure to genotoxic chemicals. Second, if the frequencies of alleles or other genetic markers differ between genotoxicant-contaminated and reference populations, associations between relative amount of DNA damage and genotype may provide evidence that these changes are due to genotoxicant-induced selection. Third, genetic analysis of gene flow may provide insight into patterns of dispersal that could obscure differences between contaminated and reference populations. In order to demonstrate the application of these ideas, three lines of research are summarized herein. The first is a series of studies that focus on radionuclide-contaminated populations of mosquitofish (Gambusia). This research identified RAPD markers that may be indicative of genetic adaptation to radionuclide stress. Relative amounts of DNA damage among genotypes presented evidence that these markers may be indicators of relative radioresistance. The second study examined DNA damage and population genetic structure in radionuclide-contaminated kangaroo rat (Dipodomys) populations. It was found that between-population differences in genetic diversity paralleled those for DNA damage and relative levels of contamination. Also, population genetic analysis indicated that there was dispersal between contaminated and reference populations, and that between-population differences in the amount of DNA damage could not be detected until this dispersal was taken into account. In the third study, populations of redbreast sunfish (Lepomis auritis) from streams contaminated with complex mixtures of industrial chemicals were examined. It was found that the genetic distances between populations within the contaminated stream corresponded with the relative magnitude of molecular and community-level effects. It was concluded that genetic ecotoxicology could make significant contributions to the fields of environmental biomonitoring and ecological risk assessment, and that integration of genotoxicology and population genetic studies would be a definite advantage toward this end.     

The DNA repair host-mediated assay as a rapid and sensitive in vivo procedure for the determination of genotoxic factors present in various organs of mice

The DNA repair host-mediated assay, in which repairable DNA damage is determined in E. coli cells present in various organs of mice exposed to genotoxic agents, was further developed to broaden the range of organs under study and to simplify the procedure of assessing differential bacterial cell survival. A pair of derivatives of E. coli K-12 strain 343/113 was constructed which differed vastly in DNA repair capacity (uvr ⁺/rec ⁺ vs uvrB/recA), as a means of assessing DNA damaging effects; furthermore, the strains differed in their ability to ferment lactose ( Lac vs Lac ⁺), so that the individual survival of both strains could be determined on a single agar medium (containing neutral red as pH indicator), on which the strains had different colony colour morphology (red, Lac ⁺ vs white, Lac ^– colonies). Finally, the strains were made streptomycin-dependent, to prevent uncontrolled growth of the bacterial cells within the various organs and also to inhibit contamination of the survival agar medium by representatives of the normal intestinal microflora.The experimental procedure consisted of injecting mixtures of stationary cells of the two strains (ca. 3–5×10⁸ viable cells per mouse) both intravenously and orally into mice, either pretreated or subsequently treated with test chemicals. Ninety minutes after injection of the bacteria, the liver, spleen, lungs, kidneys, stomach, intestine, colon, and ca. 50 l blood, were removed, suspended in buffer, homogenized, and the survival of the two strains determined on neutral red agar supplemented with streptomycin.In preliminary experiments in which the mice were treated with intraperitoneal injections of mitomycin C (0–2.0 mg per kg body weight), a dose-dependent increase in DNA damaging activity was induced in bacterial cells present in all organs tested, the lowest effects being observed in kidneys and lungs, and the highest in liver and blood. These results need further confirmation in more extensive tests, but they do nevertheless clearly indicate the possible usefulness of the DNA repair host-mediated assay as a rapid biological dose monitor for obtaining information on the genotoxic activity in vivo of compounds for which long-term mutagenicity and carcinogenicity data are not yet available.     

Appreciation of the value of different bacterial test systems for detecting and for ranking chemical mutagens

In more than thirty years of intensive research and development in the field of bacterial mutagenicity testing, a wide range of strains with various genetic endpoints has become available for routine studies. Selective genetic systems of great simplicity and handiness have been developed which assay for gene mutations, such as reversions from auxotrophy and forward mutations to antimetabolite resistance, lysogenic induction of prophages, and DNA repair. The introduction of techniques which take the mammalian metabolism into account, furthermore, makes bacterial systems potentially useful in (i) the primary identification of mutagens and non-mutagens and (ii) the quantification and ranking of relative mutagenic potency.For primary identification purposes, sensitive strains of Salmonella typhimurium LT2 and of Escherichia coli B and K-12 with altered DNA repair capacities have been constructed which demonstrate a high predictive value for many chemical mutagens (and carcinogens). Comparitive studies showed that techniques using these strains can be efficiently standardized and calibrated. Several classes of chemicals with known mutagenic properties, however, are still not detected, or underestimated, in using standard assay procedures. Examples are presented of such compounds, which can be efficiently detected as mutagens upon — slight — modifications of the experimental conditions or the DNA repair capacity of the tester strains. It appears, therefore, advisable to modify and adjust the standard testing protocol to the particular type of chemical under study and to thoroughly calibrate the system with appropriate mutagenic and non-mutagenic reference compounds.For the quantification of mutagenic potency, question remains whether bacterial systems will be of general usefulness. There are indications that the mutagenic activity of certain classes of chemicals, e.g., oxazaphosphorines, aflatoxins, nitroimidazoles, shows some proportionality with their chemical reactivity and that the relative degree of mutagenicity is similar in bacteria and in eukaryotic systems. The attractive possibility of employing in the quantification process those tester strains used for the primary identification appears, however, problematic, because (i) back mutation systems are rather specific in their response to certain mutagens and (ii) the use of strains with altered DNA repair and metabolism may lead to gross overestimations or underestimations of mutagenic potency in the corresponding wild types. More systematic studies are needed to determine the most representative genetic endpoints and genetic background under accurate determination of dose to the target molecule. The feasibility of such quantitative comparative studies has recently been demonstrated in a variety of organisms including bacteria, fungi, Drosophila, and mammalian cells in vitro and in vivo. Proposals are made to use in further comparative studies chemicals, such as procarbazine, cyclophosphamide, and ethylnitrosourea, for which data on gene mutation induction in germinal cells of mammals are available.     

A first step toward liposome-mediated intracellular bacteriophage therapy

Objectives: The emergence of antibiotic-resistant bacteria presents a severe challenge to medicine and public health. While bacteriophage therapy is a promising alternative to traditional antibiotics, the general inability of bacteriophages to penetrate eukaryotic cells limits their use against resistant bacteria, causing intracellular diseases like tuberculosis. Bacterial vectors show some promise in carrying therapeutic bacteriophages into cells, but also bring a number of risks like an overload of bacterial antigens or the acquisition of virulence genes from the pathogen.

Methods: As a first step in the development of a non-bacterial vector for bacteriophage delivery into pathogen-infected cells, we attempted to encapsulate bacteriophages into liposomes.

Results: Here we report effective encapsulation of the model bacteriophage λeyfp and the mycobacteriophage TM4 into giant liposomes. Furthermore, we show that liposome-associated bacteriophages are taken up into eukaryotic cells more efficiently than free bacteriophages.

Conclusion: These are important milestones in the development of an intracellular bacteriophage therapy that might be useful in the fight against multi-drug-resistant intracellular pathogens like Mycobacterium tuberculosis.     

Mechanisms of multistep carcinogenesis: keys to developing in vitro approaches for assessing the carcinogenicity of chemicals

The process of neoplastic development is a complex combination of genetic and non-genetic changes within a tissue. It is, therefore, not surprising that a number of mechanisms exist by which chemicals influence this process. In designing short-term tests to detect potential carcinogens, the major emphasis to date has been on genetic assays. This is justified on both theoretical and practical grounds. Substantial evidence exists to support the hypothesis that genetic damage increases the probability of tumour development. Mutational assays for gene mutations, clastogenicity and aneuploidy induction are therefore extremely important tests for carcinogens. Unfortunately, few assays exist for detecting chemicals that induce gene amplification, and thus, if these are carcinogens that specifically induce this type of genetic change, they may go undetected. Because of a variety of possible differences between a chemical's activity in vivo and in vitro, it is to be expected that chemicals that are mutagenic in vitro may not have a significant carcinogenic effect in vivo. However, the potential harm of these chemicals to man does exist and may be expressed under different conditions (e.g. species- or organ-specific carcinogenic effects) or as non-carcinogenic hazards.     

Comparative studies of ethyl methanesulfonate-induced mutations with host-mediated,dominant lethal,and cytogenetic assays

Recent reports have advocated the use of a battery of tests for evaluating the mutagenic potential of chemicals. The present study was undertaken to compare three such procedures: the host-mediated, the dominant lethal, and the in vivo cytogenetic assays, using the mutagen ethyl methanesulfonate (EMS). The relative sensitivities of the three assay procedures were determined by establishing dose-response curves and no-effect dose levels. Dominant lethal effects were not evident until a dose of 150 mg/kg was used. Cytogenetic studies indicated significant breakage of somatic cell chromosomes did not occur until a dose of 115 mg/kg was utilized. Ethyl methanesulfonate is most effective on these cells in the late S or early G₂ phases of the cell cycle. The host-mediated assay was the most sensitive of the three procedures with a statistically significant response detectable at 35 mg/kg. This difference in sensitivities may reflect a difference in repair mechanisms between microbial and mammalian cells.     

Low concentrations of permethrin and malathion induce numerical and structural abnormalities in KMT2A and IGH genes in vitro

Pesticides are commonly used worldwide and almost every human is potentially exposed to these chemicals. Exposure to pesticides such as permethrin and malathion has been associated with hematological malignancies in epidemiological studies. However, biological evidence showing if these chemicals induce genetic aberrations involved in the etiology of leukemia and lymphoma is missing. In our previous work, we have shown that a single high exposure (200 μm , 24 hours) of permethrin and malathion induce damage in genes associated with hematological malignancies in peripheral blood mononuclear cells analyzed by interphase fluorescence in situ hybridization (FISH). In the present study, we assessed by FISH whether exposure to low concentrations (0.1 μm , 72 hours) of permethrin and malathion induce aberrations in KMT2A and IGH genes, which are involved in the etiology of leukemia and lymphoma. Peripheral blood mononuclear cells were exposed to the chemicals, and damage in these genes was assessed on interphases and metaphases. We observed that both chemicals at low concentration induced structural aberrations in KMT2A and IGH genes. A higher level of damage was observed in KMT2A gene with malathion treatment and in IGH gene with permethrin exposure. We also observed numerical aberrations induced by these chemicals. The most frequent aberrations detected on interphase FISH were also observed on metaphases. Our results show that permethrin and malathion induce genetic damage in genes associated with hematological cancer, at concentrations biologically relevant. In addition, damage was observed on dividing cells, which suggests that these cells maintain their proliferation capacity in spite of the genetic damage they possess.     

Embryotoxic effects of nonylphenol and octylphenol in sea urchin <Emphasis Type="Italic">Arbacia lixula</Emphasis>

Nonylphenol (NP) and octylphenol (OP), both of which are biodegradation products of alkylphenols, are widely used in industrial applications and in some domestic products. These chemicals are found widely in surface water and aquatic sediments. We have carried out a comparative embryotoxicity analysis of the effects of increasing concentrations of NP (seven concentrations ranging from 0.937 to 18.74 μg/l) and OP (six concentrations ranging from 5 to 160 μg/l) on embryos of the sea urchin Arbacia lixula. The indicators evaluated were larval malformations, developmental arrest and embryonic/larval mortality. The results revealed that low concentrations of these chemicals (NP, OP) generally caused malformations in the skeletal system. High concentrations (18.74 μg NP/l, 160 μg OP/l) were found to inhibit the growth of embryos in the early life stages by preventing mitosis. We conclude that NP and OP present a major risk to the normal development of A. lixula at the low concentrations that have been recorded in the environment. These chemicals are therefore most likely to represent an ecological hazard at the population level given the cumulative effects of other environmental pollutants.     

Antibiotic resistance of bacterial biofilms

A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis and other components of the body's defence system. The persistence of, for example, staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is caused by biofilm-growing mucoid strains. Characteristically, gradients of nutrients and oxygen exist from the top to the bottom of biofilms and these gradients are associated with decreased bacterial metabolic activity and increased doubling times of the bacterial cells; it is these more or less dormant cells that are responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations as well as with quorum-sensing-regulated mechanisms. Conventional resistance mechanisms such as chromosomal β-lactamase, upregulated efflux pumps and mutations in antibiotic target molecules in bacteria also contribute to the survival of biofilms. Biofilms can be prevented by early aggressive antibiotic prophylaxis or therapy and they can be treated by chronic suppressive therapy. A promising strategy may be the use of enzymes that can dissolve the biofilm matrix (e.g. DNase and alginate lyase) as well as quorum-sensing inhibitors that increase biofilm susceptibility to antibiotics.     

Mutagenicity studies with praziquantel,a new anthelmintic drug: Tissue-, host-, and urine-mediated mutagenicity assays

Praziquantel, a new anthelmintic drug with activity against all species of schistosomes pathogenic to man, and against a wide range of Cestodes, was tested for mutagenic potential. For the detection of both base substitutions and frameshift mutations, Salmonella typhimurium TA 100 and TA 98 were used as tester strains. Using the plate assay with and without added S-9, host-mediated assay and urine-mediated assay without and after incubation with -glucuronidase/arylsulfatase, no mutagenic activity could be detected.     

Mutagenicity testing with Drosophila

The fruit fly, Drosophila melanogaster with its well known genetics is used to detect newly induced mutations in germ cells. Assay systems are available to determine point mutations, chromosome aberrations and nondisjunction. The sex-linked recessive lethal test is of primary practical importance, because it can detect mutagens with and without chromosome-breaking ability, which induce point mutations, small deletions or aberrations having a recessive lethal effect. For routine testing X-chromosomes are exposed to a potentially mutagenic treatment while the chromosome is in a male germ cell. In two successive crosses the treated X-chromosome is transferred to males of the F2-generation. Here, males carrying the treated chromosome can be detected based on easily scorable phenotypic characteristics (eye shape and color). The X-chromosome in F2-males is hemizygous, and therefore any recessive mutation is expressed: The presence of a recessive lethal kills all these males before they have developed into a fly, and this is easily scored as the absence of one mendelian class among the progeny.The predictability of the test for established mutagens and carcinogens is quite good. In the order of 200 compounds are known for which positive results in the sex-linked recessive lethal assay were reported. Mono- and polyfunctional alkylating agents are easily detected even if they do not break chromosomes. Frameshift mutagens can be detected, but a systematic validation of the system for this type of damage would be very desirable. Of outstanding importance is the fact that a large variety of indirect acting carcinogens are easily detected in the Drosophila in vivo assay. It is assumed that the metabolic transformation takes place within the cell subsequently tested for the presence of induced mutations (e.g., spermatids). In addition to the more indirect evidence from mutagenicity tests, information is now accumulating on the biochemical characterization of xenobiotics metabolizing systems in Drosophila and on their inducibility.An important technical problem is the selection of the route of application. Depending on the aim of the experiment and on the chemical properties of the compound under test, the route of application has to be selected with care, in order to assure that a significant exposure of the germ cells to the chemical or to its metabolites is achieved. Occasionally problems are encountered with water-insoluble compounds and the use of DMSO as solvent. These problems are currently under investigation. Possible means to increase the sensitivity of the test system for polycyclic hydrocarbons and aromatic amines are also studied. The use of Drosophila as an alternative test provides information on:(i) mutations in germ cells, (ii) in vivo activation, (iii) broad mutation spectrum (base pair substitutions, frameshifts, deletions, etc.) in one single experiment, (iv) relevant dose level which can be expected to lead to a doubling of the human spontaneous mutation frequency.The Drosophila tests have to be used in all cases in which point mutations in germ cells are considered a possible risk.     

Chronic oral LOAEL prediction by using a commercially available computational QSAR tool

In the absence of toxicological data, as it is the case for, e.g. naturally occurring substances and chemicals underlying the new European chemicals legislation, distinct tools to derive quantitative toxicological data are of particular interest with regard to risk assessment of substances humans are repeatedly exposed. The software package TOPKAT 6.2 version 3.1 (Accelrys Inc., San Diego, USA) is a commercially available tool containing a (sub)chronic oral low observed adverse level (LOAEL) prediction model constructed by using structures and LOAELs of 393 chemicals contained in publicly accessible data banks. Applying this tool, we tested the prediction of (sub)chronic LOAELS for 807 industrial chemicals (purity ≥ 95%) by comparing the predicted values with their experimental LOAELs derived from repeated dose animal experiments performed according to standard guidelines. For 460 chemicals, a prediction could not be performed because of exclusion criteria defined in the system. They had either a lower LD50 as the predicted LOAEL (n = 214) were outside the optimum prediction space which defines the domain of applicability (n = 175), were used in the training data set (n = 155), were not known to the system (n = 50) or fulfilled other criteria for data exclusion (n = 21). Of the remaining 347 substances, 34 to 62% LOAELs were predicted within a range of 1/5 and fivefold of the experimental LOAEL (factor 5), whereas 84 and 99% of the predicted LOAELs were within a range of 1/100 and 100-fold indicating high uncertainty of the prediction. Hence, a refined prediction tool is highly warranted. However, the uncertainty of the prediction could be accounted for if an additional factor of 100 is applied in addition to standard default adjustment factor of 100 which would result in an adjustment factor of 10,000 to be able to use a predicted NOAEL for risk assessment..     

In vitro neurotoxicity data in human risk assessment of polybrominated diphenyl ethers (PBDEs): Overview and perspectives

Polybrominated diphenyl ethers (PBDEs) are flame retardants routinely detected in samples of cord blood and breast milk. Concerns have been raised with regard to the toxicity of both pre- and postnatal exposures towards the developing nervous system. Although there is an increasing body of literature on the disruption of brain cell functions by certain PBDE congeners in vitro, some challenges have yet to be tackled to enable the translation of in vitro findings into their in vivo counterparts. In this paper, we review findings on the PBDE neurotoxicity in human cells and discuss the research gaps to be addressed. Moreover, we propose a scheme for the incorporation of in vitro data in human risk assessment, namely through (i) the determination of in vitro cell benchmark levels; (ii) the consideration of uncertainties in establishing equivalency between the in vitro and the in vivo tissue benchmark levels (e.g., chronic vs. acute exposure, interactions with other chemicals); and (iii) relating tissue benchmark levels to surrogate levels of internal exposure. Alongside the assessment of brain dosimetry following exposure to PBDEs, in vitro neurotoxicity data provide a unique opportunity to evaluate the risks of prenatal and early life exposures on children neurodevelopment.     

Genetic Polymorphism of Drug Metabolizing Enzymes: New Mutations in CYP2D6 and CYP2A6 Genes in Japanese

Genetic polymorphism of drug metabolizing enzymes, particularly cytochrome P450 (CYP), is an important cause of adverse drug reactions. Multiple gene mutations in CYP have been shown to be phenotype. The occurrence of genetic polymorphism has been seen in genes for CYP1A1, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A5. This review discusses the molecular mechanism of two genetic polymorphisms, debrisoquine/sparteine (CYP2D6) coumarin (CYP2A6) polymorphisms. In addition, elucidation of gene mutations of CYP2D6 and CYP2A6 in Japanese will be discussed.