Plant tissue culture as a model system for mutagenicity testing of chemicals

Two karyotypically stabilized callus strains of Crepis capillaris (2n = 7 and 2n greater than or equal to 12, respectively) were employed to illustrate the utility of plant tissue culture method for screening and analysis of cytogenetic effects of chemicals following long-term treatment. The cytogenetic analysis of callus cells revealed significant differences in the toxic and mutagenic effects of chemicals under study (2,4-D, MH, NMU and kinetin). A certain dose-response relationship (though not necessarily linear) was observed. The maximum cytogenetic activity of chemicals was associated with certain intermediate concentrations (4.5 X 10(-5) M-9 X 10(-5) M), whereas a further increase in the dose either gave rise to the opposite effects (i.e. decrease in the ana- and telo-phase aberration rates and increase in the modal karyotype frequency) or the aberration rate remained unchanged.     

Evaluation of microbial testing methods for the mutagenicity of quinoline and its derivatives

Using the mutational enhancement method and the Ames test, the mutagenicity and potential carcinogenicity of quinoline and its derivatives were determined and compared. Quinoline, 8-hydroxyquinoline, 5-hydroxyquinoline, 8-hydroxyquinoline sulfate, 6-nitroquinoline, 8-nitroquinoline, and 3-methylquinoline were mutagenic in the Ames direct plating test on TA 98 and TA 100 with activating system (S-9) from the rat liver. These compounds were not mutagenic in the mutational enchancement test onEscherichia coli HCR+ strain. 5,7-Dichloro-8-hydroxyquinoline, isoquinoline, and 2-chloroquinoline were nonmutagenic without or with S-9 in both the Ames and mutational enhancement test system. The compounds chloroquine, primaquine diphosphate, quinine hydrobromide, quinine hydrochloride, quinine lactate, quinine urea hydrochloride, quinine ethylcarbonate, quinine dihydrochloride, beta quinine quinine valerate, and quinine glycerophosphate were nonmutagenic with and without S-9 in the Ames test but mutagenic (20–60 g/ml) in the mutational enhancement test method onEscherichia coli HCR+ strains. The observations reported here point out that the Ames test responds negatively to several quinoline derivatives that are positive in the mutational enchancement test method.     

Reconsidering environmental effects assessment of chemicals: Proposal for a dynamic testing strategy

Certain substances may be hazardous to ecosystems. To be able to preserve the structures and functions of ecosystems, knowledge is required to qualify and quantify such hazards. To this end, biotests are indispensable tools. For the development and/or choice of biotests, special attention has to be drawn to conflicts between scientific demands and practical constraints. From a purely scientific point of view, experiments should be designed to maximise the ecological relevance of the obtained results. However, this often collides with the limited resources (budget, time, manpower) available. Furthermore, societal issues (e.g. animal welfare) have to be taken into account. Thus, it is necessary to develop a scientifically sound testing approach that avoids unnecessary animal testing, keeps the costs low, and can be performed within a short timeframe. The different perspectives of ecology, environmental toxicology, and environmental chemistry should be integrated into a balanced ecotoxicological approach. Accordingly, we propose a dynamic testing strategy, which is adapted to the substance (or substance group) in question and its mode(s) of action.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Topic analysis: an objective measure of the consultation and its application to computer assisted consultations.

A simple method has been developed to classify the verbal interaction during medical consultations in terms of the relative proportions of medical and social content and the initiator of conversational topics discussed. The method has been applied to video tape recordings of three doctors'' consultations with and without a computer present to classify and compare the items discussed. Actual computer use has been shown to have a medical effect on the consultations (p less than 0.05) and to increase the proportion of topics initiated by the doctor (p less than 0.001). Although this was largely accounted for by the massive increase in doctor initiated medical items resulting directly from computer use, there was evidence that, for two of the doctors, these topics were replacing some of the normal social and patient initiated medical exchanges.     

Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae

Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues. At least 30% of all proteins are thought to contain covalently bound phosphate. Despite the importance and widespread occurrence of this modification, identification of sites of protein phosphorylation is still a challenge, even when performed on highly purified protein. Reported here is methodology that should make it possible to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment. Proteins are digested with trypsin and the resulting peptides are then converted to methyl esters, enriched for phosphopeptides by immobilized metal-affinity chromatography (IMAC), and analyzed by nanoflow HPLC/electrospray ionization mass spectrometry. More than 1,000 phosphopeptides were detected when the methodology was applied to the analysis of a whole-cell lysate from Saccharomyces cerevisiae. A total of 216 peptide sequences defining 383 sites of phosphorylation were determined. Of these, 60 were singly phosphorylated, 145 doubly phosphorylated, and 11 triply phosphorylated. Comparison with the literature revealed that 18 of these sites were previously identified, including the doubly phosphorylated motif pTXpY derived from the activation loop of two mitogen-activated protein (MAP) kinases. We note that the methodology can easily be extended to display and quantify differential expression of phosphoproteins in two different cell systems, and therefore demonstrates an approach for "phosphoprofiling" as a measure of cellular states.     

Sulfation of environmental estrogen-like chemicals by human cytosolic sulfotransferases

To investigate whether sulfation, a major Phase II detoxification pathway in vivo, can be employed as a means for the inactivation/disposal of environmental estrogens, recombinant human cytosolic sulfotransferases were prepared and tested for enzymatic activities with bisphenol A, diethylstilbestrol, 4-octylphenol, p-nonylphenol, and 17alpha-ethynylestradiol as substrates. Of the seven recombinant enzymes examined, only SULT1C sulfotransferase #1 showed no activities toward the environmental estrogens tested. Among the other six sulfotransferases, the simple phenol (P)-form phenol sulfotransferase and estrogen sulfotransferase appeared to be considerably more active toward environmental estrogens than the other four sulfotransferases. Metabolic labeling experiments revealed the sulfation of environmental estrogens and the release of their sulfated derivatives by HepG2 human hepatoma cells. Moreover, sulfated environmental estrogens appeared to be incapable of penetrating through the HepG2 cell membrane.     

Legislative and technical aspects of mutagenicity testing.

A brief account is given of the history of the legislative acts that give responsibility to the U.S. Food and Drug Administration (FDA) for ensuring the safety of foods, drugs, and cosmetics. Within the present legislative framework the FDA has the authority to impose regulations which are designed to ensure the safety of all foods, drugs, and cosmetics. The existing legislative authority is adequate for this purpose; however, the difficulty lies instead with technology and the inadequacy of scientific perspective in the emerging area of mutagenicity testing. Earlier efforts in development of mutagenicity screening systems culminated only a few years ago in the proposal to use the host-mediated assay, somatic cell cytogenetics, and dominant lethal tests collectively. Subsequent research efforts indicated that there were serious practical and scientific deficiencies in using this approach. More recently a new proposal, the tier system, has been suggested as an alternative measure. The proposed tier system at FDA consists of three testing levels of increasing complexity. The first tier is an initial screening effort using techniques having maximum sensitivity that are also useful for large-scale, rapid testing. The second tier is designed to identify and confirm that the presumptive mutagens detected in the first tier are truly mutagenic for higher organisms, most especially, for mammals. The third tier would be devoted to explicit genetic tests in mammals designed to ascertain the imposed risk to man by the introduction of a mutagen in our environment. The FDA is currently involved in a number of research activities in the area of mutagenicity safety screening which will explore the adequacies and possible deficiencies of the tier system approach. These efforts are described for our in-house activities, our contract activities, and our cooperative and collaborative activities with other government agencies and institutions.     

Incorporation of mammalian metabolism into mutagenicity testing

In the 1950's and 1960's it became obvious that many chemicals in daily use were mutagenic or carcinogenic, but there seemed to be little relation between the two activities. As scientists were debating the cause of this discrepancy, it was hypothesized that mammalian metabolism could form highly reactive intermediates from rather innocuous chemicals and that these intermediates could react with DNA and were mutagenic. This commentary presents the historical development of metabolic activation in mutagenicity tests, beginning with Udenfriend's hydroxylation system, which mimics aspects of mammalian metabolism in a purely chemical mixture, and extending through procedures that moved closer and closer to incorporating actual mammalian metabolism into the test systems. The stages include microsomal activation systems, host-mediated assays, incorporation of human P450 genes into the target cells or organisms, and detecting mutations in single cells in vivo. A recent development in this progression is the insertion of recoverable vectors containing mutational targets into the mammalian genome. Since the target genes of transgenic assays are in the genome, they are not only exposed to active metabolites, but they also undergo the same repair processes as endogenous genes of the mammalian genome.     

Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing

Individual chromosomes can be identified by means of in situ hybridization with DNA probes for chromosome-specific repetitive sequences. The efficiency and sensitivity of the method are strictly dependent on the characteristics of the probes and the experimental conditions. Using three probes with different copy numbers, we demonstrated that the target chromosomes can be visualized in interphase when the homologous sequences are repeated at least 50 times.Possible applications of interphase analysis to clinical cytogenetics and mutagenicity testing are discussed.     

Disruption of androgen receptor signaling in males by environmental chemicals

Androgen-disruptors are environmental chemicals in that interfere with the biosynthesis, metabolism or action of endogenous androgens resulting in a deflection from normal male developmental programming and reproductive tract growth and function. Since male sexual differentiation is entirely androgen-dependent, it is highly susceptible to androgen-disruptors. Animal models and epidemiological evidence link exposure to androgen disrupting chemicals with reduced sperm counts, increased infertility, testicular dysgenesis syndrome, and testicular and prostate cancers. Further, there appears to be increased sensitivity to these agents during critical developmental windows when male differentiation is at its peak. A variety of in vitro and in silico approaches have been used to identify broad classes of androgen disrupting molecules that include organochlorinated pesticides, industrial chemicals, and plasticizers with capacity to ligand the androgen receptor. The vast majority of these synthetic molecules act as anti-androgens. This review will highlight the evidence for androgen disrupting chemicals that act through interference with the androgen receptor, discussing specific compounds for which there is documented in vivo evidence for male reproductive tract perturbations. This article is part of a Special Issue entitled 'Endocrine disruptors'.     

Human metabolic interactions of environmental chemicals

Investigations utilizing recombinant human xenobiotic-metabolizing enzymes as well as human hepatocytes have revealed a number of interactions not only between different environmental chemicals (ECs) but also between ECs and endogenous metabolites. Organophosphorus insecticides (OPs) are potent inhibitors of the human metabolism of carbaryl, carbofuran, DEET and fipronil, as well as the jet fuel components, nonane and naphthalene. OPs are potent irreversible inhibitors of testosterone metabolism by cytochrome P450 (CYP) 3A4 and of estradiol metabolism by CYP3A4 and CYP1A2. All of these CYP inhibitions are believed to be due to the release of reactive sulfur during CYP-catalyzed oxidative desulfuration. It has also been shown that the esterase(s) responsible for the initial step in permethrin metabolism in human liver is inhibited by both chlorpyrifos oxon and carbaryl. A number of pesticides, including chlorpyrifos, fipronil and permethrin, and the repellent, DEET, have been shown to be inducers of CYP isoforms in human hepatocytes, with fipronil being the most potent. Several agrochemicals, including fipronil and the pyrethroids, permethrin and deltamethrin, show toxicity toward human hepatocytes with fipronil being the most potent in this regard. Endosulfan-alpha, which has shown promise as a model substrate for phenotyping CYP3A4 and CYP2B6 in human liver microsomes, is also an inducer of CYP2B6, acting through the PXR receptor.     

Carcinogenicity and mutagenicity testing, then and now.

Cancer is a dread disease worldwide. Mortality of individuals suffering from cancer is high, despite the current improved methods of precocious detection, surgery and therapy. Prevention of cancer is the recognized goal of many activities in cancer research. This aim was recognized early to involve the bioassay of environmental chemicals or mixtures. The first such study involved application of coal tar to the ear of rabbits, and later on to the skin of mice. Subsequently, laboratory rats were introduced, and hamsters were utilized as a substitute for the unwieldy tests in rabbits. Investigators also became concerned with the mechanisms of carcinogenesis, and more definitive approaches to carcinogen bioassay in laboratory animals, as possible indicators of cancer risk in humans. These tests were expensive and lengthy, and did not serve the important purpose of accurately measuring risk of cancer to humans. Once it was realized that DNA and the genetic apparatus might be a key target, rapid bioassays in bacterial and mammalian cell systems were introduced successfully. Thus, batteries of tests are now available to detect effectively human cancer risks, and provide novel approaches to determine the underlying mechanisms, as a sound basis for cancer prevention.