Animal models for Francisella tularensis and Burkholderia species: scientific and regulatory gaps toward approval of antibiotics under the FDA Animal Rule.

The development and regulatory approval of medical countermeasures (MCMs) for the treatment and prevention of bacterial threat agent infections will require the evaluation of products in animal models. To obtain regulatory approval, these models must accurately recapitulate aspects of human disease, including, but not necessarily limited to, route of exposure, time to disease onset, pathology, immune response, and mortality. This article focuses on the state of animal model development for 3 agents for which models are largely immature: Francisella tularensis, Burkholderia mallei, and Burkholderia pseudomallei. An overview of available models and a description of scientific and regulatory gaps are provided.

EuroFIR eBASIS: application for health claims submissions and evaluations.

BACKGROUND: The European Food Information Resource (EuroFIR) network has established the eBASIS (Bioactive Substances in Food Information System) online food composition and biological effects database for plant-derived bioactive compounds (phytochemicals). On the basis of submitted evidence, the European Food Safety Authority (EFSA) expert panel on Dietetic Products, Nutrition and Allergies assesses whether claims made under articles 13.1, 13.5 or 14 of the Regulation (EC) 1924/2006, which governs the use of nutrition and health claims on foods, are scientifically justified. This report evaluates the eBASIS biological effects database in the preparation and evaluation of health claims dossiers. METHODS: The eBASIS biological effects database is a compilation of expert-evaluated data extracted from the literature, prioritizing human intervention studies to investigate health effects of phytochemicals. Currently included are >750 records from 445 studies providing data on 56 validated biomarkers, mainly relating to cardio-metabolic and bone health outcomes. The data cover 144 bioactive compounds from 17 compound classes. Using the EFSA Register of Questions and the database of general function health claims, we identified claims relating to phytochemicals made under articles 13.1, 13.5 and 14 and compared them with the eBASIS database to identify overlap between them. RESULTS: The EFSA online health claims database contains 4240 submissions under article 13.1, of which 2157 pertain to plants or plant-based bioactive compounds; 496 of these relate to plants or bioactive compounds included in the eBASIS biological effects database. Out of the 18 current 13.5 'new function' claims on EFSA's register of questions, 7 are for plants or plant-based bioactive compounds, of which 6 are included in eBASIS. Of the 222 defined article 14 claims, 21 pertain to plants or plant-based bioactive compounds, of which 19 are in eBASIS. CONCLUSIONS: There is extensive overlap between eBASIS and the submitted health claims that relate to plant-based bioactive compounds. EuroFIR eBASIS is a useful tool for regulators to independently check completeness of health claims applications relating to phytochemicals and is a potentially valuable resource to assist claimants in the compilation of dossiers on functional foods and health claims.

Evaluation of certain food additives and contaminants.

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, including flavouring agents, with a view to recommending acceptable daily intakes (ADIs) and to preparing specifications for identity and purity. The Committee also evaluated the risk posed by two food contaminants, with the aim of advising on risk management options for the purpose of public health protection. The first part of the report contains a general discussion of the principles governing the toxicological evaluation and assessment of intake of food additives (in particular flavouring agents) and contaminants. A summary follows of the Committee's evaluations of technical, toxicological and intake data for certain food additives (acidified sodium chlorite, asparaginase from Aspergillus oryzae expressed in Aspergillus oryzae, carrageenan and processed Eucheuma seaweed, cyclotetraglucose and cyclotetraglucose syrup, isoamylase from Pseudomonas amyloderamosa, magnesium sulfate, phospholipase A1 from Fusarium venenatum expressed in Aspergillus oryzae, sodium iron(III) ethylenediaminetetraacetic acid (EDTA) and steviol glycosides); eight groups of related flavouring agents (linear and branched-chain aliphatic, unsaturated, unconjugated alcohols, aldehydes, acids and related esters; aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally related substances; simple aliphatic and aromatic sulfides and thiols; aliphatic acyclic dials, trials and related substances; aliphatic acetals; sulfur-containing heterocyclic compounds; aliphatic and aromatic amines and amides; and aliphatic alicyclic linear alpha, beta -unsaturated di- and trienals and related alcohols, acids and esters); and two food contaminants (aflatoxin and ochratoxin A). Specifications for the following food additives were revised: maltol and ethyl maltol, nisin preparation, pectins, polyvinyl alcohol, and sucrose esters of fatty acids. Specifications for the following flavouring agents were revised: maltol and ethyl maltol, maltyl isobutyrate, 3-acetyl-2,5-dimethylfuran and 2,4,5-trimethyl-delta-oxazoline (Nos 1482, 1506 and 1559), and monomenthyl glutarate (No. 1414), as well as the method of assay for the sodium salts of certain flavouring agents. Annexed to the report are tables summarizing the Committee's recommendations for intakes and toxicological evaluations of the food additives and contaminants considered.

A safe strategy for addition of vitamins and minerals to foods.

Addition of vitamins and minerals to foods must be done without health risk to any consumer group. International expert groups have aimed at establishing tolerable upper intake levels (ULs) for vitamins and minerals although lack of solid data on their safety is a major obstacle to this work. In this paper, we summarize the existing ULs and suggest the use of guidance levels (GLs) set by others and temporary guidance levels (TGLs) proposed here, whenever no consensus UL has been established for adults. We suggest the use of body surface area ratios to establish similar levels for younger age groups. The levels are applied in a model for calculation of safe fortification levels for all ages. We have estimated the upper 95(th) percentile intake of vitamins and minerals from food in various Danish age and gender groups and suggest that a daily multivitamin-mineral pill is included in the calculation of total dietary intake levels of all vitamins and minerals. By subtracting this dietary intake level from the UL, GL or TGL, we calculate the amount that can be safely used for fortification. Since safety must be assured for all age groups, the smallest difference relative to energy intake calculated for any age group is proposed as the maximal allowance (MA) for fortification with each nutrient. We suggest that the MA should be expressed in weight units per energy unit in order to distribute it equally between potentially fortifiable food groups according to their usual contribution to total energy intakes.

Applicability of the CALUX bioassay for screening of dioxin levels in human milk samples.

The CALUX (chemically activated luciferase expression) bioassay based on rat hepatoma (H4IIE) cells is a sensitive assay for the detection of Ah receptor agonists like 2,3,7,8-substituted chlorinated dibenzo-p-dioxins and dibenzofurans and related PCBs. In this paper, the assay was optimized and applied for monitoring levels of dioxins in human milk samples. Combination effects of dioxin-like compounds were evaluated by testing potential mechanisms of interaction between seven of the major dioxin-like compounds in human milk using the isobole method. Results showed that the compounds acted additively, indicating that the usual assumption of additivity in the risk assessment process is valid. In general the relative potencies (REPs) of the single agents were in accordance with their TEFs assigned by the World Health Organisation, except for the mono-ortho-substituted PCB118 that had a 40-fold lower REP in CALUX. The total dioxin-like activity was determined in 16 Danish human milk samples and was in the range 20.5-55.8 pg TEQ g(-1) fat. These values were compared with TEQs obtained from GC/MS analysis (range 14.8-43.6 pg TEQ-g(-1) fat) that overall were a little lower than CALUX TEQs. The results obtained with the bioassay when testing milk extracts fractionated into dioxins/furans, non-ortho PCB and mono/di-ortho PCB fractions indicated that the correlation between the bioassay and the chemical analyses depends primarily on the Ah receptor activity observed in the mono/di-ortho PCB fraction.

Current risk assessment approaches in different countries.

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) exist as complex mixtures in environmental and biological samples. There is sufficient evidence that the toxic congeners share a common mode of action, involving binding to the Ah-receptor. Toxic equivalency factors (TEFs) and chemical residue data are used to calculate toxic equivalent (TEQ) concentrations in environmental samples, foods, animal and human tissues. Two different approaches have been used in the risk assessments of PCDDs, PCDFs and dioxin-like PCBs. WHO and most countries outside the USA have derived Tolerable Daily (or weekly) Intakes (TDI) in the order of 1-10 pg per kg of body weight for TCDD or TEQs based on data from rodent carcinogenicity studies. These countries have assumed the existence of a threshold dose for the carcinogenicity of dioxins, while US EPA and USFDA have used probabilistic estimates of cancer potency, treating cancer as a non-threshold effect and using a descriptor that addresses upper bound risk, the Risk Specific Dose (RsD). In the USA and other countries there is a growing concern over the non-cancer effects of dioxin-like compounds. In general, the various risk assessments have identified groups of the population that are at particular high risks and all have stressed the urgent need to reduce the sources of the environmental contamination with these compounds to the lowest possible.

Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro.

Many pesticides are able to block or activate the steroid hormone receptors and/or to affect the levels of sex hormones, thereby potentially affecting the development or expression of the male and female reproductive system or both. This emphasizes the relevance of screening pesticides for a wide range of hormone-mimicking effects. Twenty-two pesticides were tested for their ability to affect CYP19 aromatase activity in human placental microsomes using the classical [(3)H](2)O method. Prochloraz, imazalil, propioconazole, fenarimol, triadimenol, triadimefon (all fungicides), and dicofol (an acaricide) gave rise to a statistically significant inhibition of aromatase activity. The IC(50)s of prochloraz, imazalil, propioconazole fenarimol, triadimenol, and triadimefon were calculated from dose-response curves to be 0.04, 0.34, 6.5, 10, 21 and 32 microM, respectively. The IC(50) of dicofol was greater than 50 microM. The positive control 4-hydroxyandrostendione (1 microM) caused an inhibition of aromatase activity by 74%. The compounds, which did not affect the aromatase activity, were bromopropylate, chlorfenvinphos, chlorobenzilate, chlorpyrifos, diuron, heptachlor, iprodion, linuron, pentachlorphenol, procymidon, propyzamide, quintozen, tetrachlorvinphos and tetradifon. With the purpose of comparing the results for fenarimol obtained with the microsomal system with data from an intact cell system, an aromatase assay based on JEG-3 cells was established. 4-Hydroxyandrostendione (1 microM) inhibited the aromatase activity in JEG-3 cells by 94%. The IC(50) for fenarimol in this system was 2 microM, slightly lower than that observed in the microsomal system. For the first time, fenarimol has been demonstrated to inhibit aromatase activity in human tissues and, furthermore, propioconazole, triadimefon, and triadimenol were identified as weak aromatase inhibitors. In conclusion, seven out of 22 tested pesticides turned out to be weak to moderate aromatase inhibitors in vitro, indicating the relevance of elucidating the endocrine effects in vivo of these- compounds.

Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro.

Nine structurally different polycyclic aromatic hydrocarbons (PAHs) were tested for their ability to either agonize or antagonize the human androgen receptor (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector. Benz[a]anthracene (B[a]A), benzo[a]pyrene (B[a]P), fluoranthene, chrysene and 7,12-dimethylbenz[a]anthracene (DMBA) were acting as antiandrogens in vitro, resulting in IC(50) values of 3.2, 3.9, 4.6, 10.3 and 10.4 microM, respectively. Only at the highest concentration tested (10 microM), a slight inhibitory effect by pyrene, phenanthrene, and anthracene was observed. In contrast, dibenzo[a,h]anthracene (DB[a,h]A) gave rise to an agonistic effect, which was added upon the effect of the androgen receptor agonist R1881 (0.1 nM). The antiandrogenic responses by PAHs (10 microM) were found to be fully reversible, determined in the presence of increasing concentrations of R1881. No cytotoxic effects of the tested compounds were observed as determined either by metabolic reduction using AlamarBlue (up to 20 microM) or determined in cells transfected with a constitutively active hAR (up to 10 microM). The well-known ability of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2,3,7,8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB[a,h]A, B[a]P, Chrysene, B[a]A and DMBA gave rise to a 4.5-fold induction of luciferase activity at 0.03, 0.4, 0.89, 3.06, and 9.27 microM, respectively, whereas fluoranthene, pyrene, phenanthrene and anthracene were without effect. In conclusion, no clear correlation between the antiandrogenic effects and the Ah receptor activation in vitro was seen. However, the Ah receptor agonists containing four or five aromatic rings (i.e. B [a] A, B [a] P, chrysene, DMBA) appeared to be the most potent antiandrogens (with the exception of DB [a, h] A), whereas those not able to activate the Ah receptor containing three or four aromatic rings (i.e. pyrene, phenanthrene, anthracene) displayed either very weak or no antiandrogenic effect at concentrations up to 10 microM (with the exception of fluoranthene which blocked the hAR at lower concentrations, but did not activate the Ah receptor).

Dioxins: WHO's tolerable daily intake (TDI) revisited.

In December 1990, the World Health Organization (WHO) established a tolerable daily intake (TDI) of 10 pg/kg b.w. (body weight) for TCDD, based on liver toxicity, reproductive effects and immunotoxicity in experimental animals, and making use of kinetic data in humans and experimental animals. Since then new epidemiological and toxicological data have emerged, in particular with respect to neurodevelopmental and endocrine effects of dioxin. Therefore, the European Centre for Environment and Health of the World Health Organization (WHO-ECEH) and the International Programme on Chemical Safety (IPCS) jointly organized a consultation on the "Assessment of the health risk of dioxins: re-evaluation of the TDI", May 1998, Geneva, Switzerland. The participants discussed the health risks for infants, cancer and non-cancer endpoints in humans and animals, mechanistic aspects, kinetic behaviour, modelling, exposure, and the applicability of the toxic equivalency (TEQ) concept. For the health risk assessment of dioxin-like compounds, the WHO Consultation focused on the most sensitive effects that are considered adverse (hormonal, reproductive and developmental effects) seen at low doses in animal studies (rats and monkeys). Human daily intakes corresponding with body burdens similar to those associated with adverse effects in animals could be estimated to be in the range of 14-37 pg/kg b.w./day. To arrive at a TDI expressed as TEQ, a composite uncertainty factor of 10 was recommended. By applying this uncertainty factor a TDI range of 1-4 pg TEQs/kg body weight was established. An extensive executive summary of the results of this WHO Consultation with all the underlying background documents will be published in Food Additives and Contaminants (in press).

Lack of oestrogenic effects of food preservatives (parabens) in uterotrophic assays.

The oestrogenic activity of the parabens, methyl-, ethyl- and propyl p-hydroxybenzoate, widely used as antimicrobials in food, and butyl p-hydroxybenzoate, which is used in cosmetic products, and their shared main metabolite p-hydroxybenzoic acid was investigated in a mouse uterotrophic assay. Immature B6D2F1 mice were treated with oral or subcutaneous doses of the test compounds for three consecutive days. p-Hydroxybenzoic acid and butyl p-hydroxybenzoate were also tested by the subcutaneous route in a rat uterotrophic assay. A significant increase in the uterus weight at day 4 was considered an oestrogenic effect. In the mouse assay, none of the compounds tested produced any oestrogenic response at dose levels up to 100mg/kg body weight per day, for ethyl p-hydroxybenzoate even at a dose level of 1000mg/kg body weight per day. In immature Wistar rats, subcutaneous administration of butyl p-hydroxybenzoate produced a weak oestrogenic response at 600mg/kg body weight per day.

Report of workshop on the significance of excursions of intake above the ADI.

The acceptable daily intake (ADI) for humans was originally developed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and defined as "an estimate of the amount of a food additive, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable health risk." JECFA has not provided any firm guidance on how to evaluate excursions of intake above the ADI, but WHO in 1987 stated that "because in most cases, data are extrapolated from life-time animal studies, the ADI relates to life-time use and provides a margin of safety large enough for toxicologists not to be particularly concerned about short-term use at exposure levels exceeding the ADI, providing the average intake over longer periods of time does not exceed it." In discussing short-term intakes in excess of recommended limits, JECFA in 1989 concluded that short-term exposures to levels exceeding the provisional tolerable weekly intake (PTWI) for a contaminant is not a cause of concern, provided the individual's intake averaged over longer periods of time does not exceed the level set. JECFA also stated that it was impossible to make a generalization concerning the length of time during which intakes in excess of the PTWI would be toxicologically detrimental. Any detrimental effect would depend upon the nature of the toxicity and the biological half-life of the chemical concerned. JECFA considered intakes of food additives in excess of the ADI less likely to occur and easier to control than in the case of contaminants which are allocated either a PTWI or a tolerable daily intake (TDI). The ILSI Europe Acceptable Daily Intake Task Force together with the Food Chemical Intake Task Force initiated a workshop which took place April 21-23, 1998, in Milan, Italy, in order to help identify what information would be needed, with what precision, and what is already available to evaluate the significance of excursions of intake above the ADI. The specific aims of the workshop were to address the following questions: By how much can the ADI be exceeded? For how long can excursions above the ADI be tolerated with respect to chronic toxicity, accumulation, and mechanisms of toxicity? What methods should be used to estimate intakes so that the estimates are relevant to the ADI? Do the same principles apply to contaminants that have TDI or PTWI values?

Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals.

Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were cotransfected with the human androgen receptor expression vector and the mouse mammary tumour virus (MMTV)2-luciferase vector using the new nonliposomal transfection reagent FuGene. Stimulation of the cells for 24 h with the synthetic androgen receptor agonist, R1881 (10 nM), resulted in a 30- to 60-fold induction of luciferase activity. The classical antiandrogenic compounds hydroxy-flutamide, bicalutamide, spironolactone, and cyproterone acetate together with the pesticide(metabolite)s, vinclozolin, p,p'-DDE, and procymidone all potently inhibited the response to 0.1 nM R1881. Compared to the traditional calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands-on laboratory time. This assay is a powerful tool for the efficient and accurate determination and quantification of the effects of antiandrogens on reporter gene transcription. To extend the application of FuGene, the reagent was shown to be superior compared to Lipofectin for transfecting MCF7 human breast cancer cells with an estrogen response element-luciferase vector. Thus, FuGene may prove to be valuable in diverse reporter gene assays involving transient transfections for screening of potential endocrine disruptors for (anti)androgenic and (anti)estrogenic properties.

Screening of selected pesticides for oestrogen receptor activation in vitro.

Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an MCF7 cell proliferation assay and a Yeast Oestrogen Screen. The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 microM induces a 2.0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone). The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor. Several pesticides did not have any effect on the proliferation response after 6 days of exposure, including: chlorpyrifos, diuron, iprodion, linuron, pentachlorphenol, prochloraz, propioconazol, propyzamine, quintozen, tetrachorvinphos and tetradifon. Some pesticides resulted in a negligible proliferation response, which was not statistically significant under the present experimental conditions. These were: bromopropylate, chlorfenvinphos, chlorobenzilate, dicofol, heptachlor, and imazalil. Fenarimol and dicofol also gave rise to a positive oestrogenic response in yeast cells transfected with the oestrogen receptor alpha, whereas the remaining compounds resulted in a negative response due either to biological inactivity or cytotoxocity to the yeast cells. The EC50 for fenarimol was estimated to be 13 microM in the yeast cells, compared with an EC50 of 3 microM in the MCF7 cells, indicating higher sensitivity of the latter assay. No in vivo data for fenarimol, triadimefon or triadimenol have previously been published that support oestrogenic activity in the intact animal. Thus, from the present results we suggest that oestrogen receptor activation may not be an important mode of action for these compounds. The need to include at least two bioassays in a screening procedure and for combining in vitro and in vivo data is emphasized.

Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife.

An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in vivo evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties.

Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay.

A newly developed recombinant yeast strain, in which the human estrogen receptor has been stably integrated into the genome of the yeast, was used to gain information on the estrogenic activity of a large series of dietary flavonoids. Among 23 flavonoids investigated, 8 were found to markedly stimulate the transcriptional activity of the human estrogen receptor in the yeast assay increasing transcriptional activity 5-13-fold above background level, corresponding to EC50 values between 0.1 and 25 microM. Five compounds increased the transcriptional activity 2-5-fold over the control, with EC50 values ranging from 84 to 102 microM, whereas the remaining flavonoids were devoid of activity. The most potent flavonoid estrogens tested were naringenin, apigenin, kaempferol, phloretin, and the four isoflavonoids equol, genistein, daidzein, and biochanin A. With the exception of biochanin A, the main feature required to confer estrogenicity was the presence of a single hydroxyl group in the 4'-position of the B-ring of the flavan nucleus, corresponding to the 4-position on phloretin. The estrogenic potency of the flavonoids was found to be 4 000-4 000 000 times lower than that observed for 17beta-estradiol, when compared on the basis of EC50 values. The estrogenic activity of the dietary flavonoids was further investigated in estrogen-dependent human MCF7 breast cancer cells. In this system several of the flavonoids were likewise capable of mimicking natural estrogens and thereby induce cell proliferation. Similar structural requirements for estrogenic activity were found for the two assays. The present results provide evidence that several of the flavo-estrogens possess estrogenic properties comparable in activity to the well-established isoflavonoid estrogens. The use of Alamar Blue, a vital dye which is metabolically reduced by cellular enzymes to a fluorescent product, was found to greatly simplify the MCF7 cell-based estrogen screen, making this mammalian assay applicable as a large-scale screening tool for estrogenic compounds.

Microsomal metabolism and activation of the environmental carcinogen 2-amino-3-methyl-9H-pyrido[23-b]indole.

2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) is a mutagenic and carcinogenic heterocyclic amine formed as a pyrolysis product during cooking of food and combustion of tobacco. Hepatic microsomes from PCB-induced rats metabolized MeA alpha C to four products, of which three were non-mutagenic and one was mutagenic without S9 activation. The three non-mutagenic products, which accounted for 83% of the metabolism of MeA alpha C, were characterized by mass spectrometry and NMR spectroscopy as 6-hydroxy-MeA alpha C, 7-hydroxy-MeA alpha C and 3-hydroxy-methyl-A alpha C. The mutagenic metabolite, accounting for 17% of the metabolism of MeA alpha C, was characterized as N2-hydroxy-MeA alpha C by comparison with the HPLC retention time and UV spectrum of N2-hydroxy-MeA alpha C obtained by chemical synthesis. N2-Hydroxy-MeA alpha C was very reactive and a part of it bound covalently to microsomal proteins during incubation and a part was degraded to other products during incubation or chromatography. N2-Hydroxy-MeA alpha C was mutagenic in Salmonella typhimurium TA98 without metabolic activation, resulting in 5070 revertants/microgram, which was > 20 times the specific mutagenic activity of the parent compound.

City air pollution of polycyclic aromatic hydrocarbons and other mutagens: occurrence, sources and health effects.

The presence of polycyclic aromatic hydrocarbons (PAH), mutagens and other air pollutants was investigated in a busy street in central Copenhagen and in a park area adjacent to the street. The winter concentration of benzo(a)pyrene was 4.4 +/- 1.2 ng/m3 in the street air and 1.4 +/- 0.6 ng/m3 in the city park. The atmospheric concentrations of PAH decreased in the order of: street > city background air approximately suburbs > village > open land. The traffic contribution of PAH to street air was estimated to be 90% on working days and 60% during weekends and its contribution to city background air was estimated to be 40%. Four different approaches to evaluate the health effects are discussed. The direct effect of PAH air pollution, and other mutagens, is considered to be a maximum of five lung cancer cases each year out of one million people.

Male reproductive health and environmental xenoestrogens.

Male reproductive health has deteriorated in many countries during the last few decades. In the 1990s, declining semen quality has been reported from Belgium, Denmark, France, and Great Britain. The incidence of testicular cancer has increased during the same time incidences of hypospadias and cryptorchidism also appear to be increasing. Similar reproductive problems occur in many wildlife species. There are marked geographic differences in the prevalence of male reproductive disorders. While the reasons for these differences are currently unknown, both clinical and laboratory research suggest that the adverse changes may be inter-related and have a common origin in fetal life or childhood. Exposure of the male fetus to supranormal levels of estrogens, such as diethlylstilbestrol, can result in the above-mentioned reproductive defects. The growing number of reports demonstrating that common environmental contaminants and natural factors possess estrogenic activity presents the working hypothesis that the adverse trends in male reproductive health may be, at least in part, associated with exposure to estrogenic or other hormonally active (e.g., antiandrogenic) environmental chemicals during fetal and childhood development. An extensive research program is needed to understand the extent of the problem, its underlying etiology, and the development of a strategy for prevention and intervention.