Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples.

The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, species-specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.

Detection of carcinogenic aromatic amines in the urine of non-smokers.

Smoking is thought to be one of the most important anthropogenic risk factors involved in the development of urinary bladder cancer in humans. Tobacco smoke contains a complex mixture of chemicals including potent carcinogens such as aromatic amines. In the present study the amounts of several freebase aromatic amines including the potent carcinogens 2-aminonaphthalene and 4-aminobiphenyl have been analyzed in the urine of 48 German urban living smokers and non-smokers. The results indicate that (i) both groups excrete the identical set of four aromatic amines; (ii) smokers excrete approximately twice the total amount of these amines, but similar amounts of 2-aminonaphthalene and 4-aminobiphenyl are found in non-smokers; and (iii) the excreted aromatic amines are decomposed in the urine within a few hours thus, explaining why aromatic amines are difficult to detect in this matrix. Their decomposition could be prevented by adding small amounts of p-toluidine to the freshly collected urine. Unlike smokers the origin of aromatic amines detected in the urine of non-smokers is at present unknown. Based on the cotinine levels found in the urine of non-smokers environmental tobacco smoke can be excluded as a major source of aromatic amines. In addition, neither diesel exhaust-related nitroarenes nor the corresponding amino-derivatives, to which they may be metabolically converted, were found. The detected urinary levels of aromatic amines arising from sources other than tobacco smoke or diesel exhaust may play a role in the bladder cancer etiology of non-smokers.

Profile of urinary phenanthrene metabolites in smokers and non-smokers.

Phenanthrene metabolites (phenols and dihydrodiols) and 1-hydroxypyrene excreted in the 24-h urine of smokers, non-smokers and lung cancer patients, who after heavy smoking became light smokers, were determined and compared. In contrast to 1- hydroxypyrene, no significant differences of the absolute amounts of phenanthrene metabolites were found between smokers and non-smokers. A ratio phenanthrene metabolites/l-hydroxypyrene of 10.4 was observed for non-smokers and 9.9 for lung cancer patients, but 4.2 for smokers. Significantly different ratios for the regiospecific oxidation of phenanthrene were found for smokers when compared with non-smokers (1,2-oxidation vs 3,4-oxidation was 1.45 in the case of smokers, but 2.34 in the case of non-smokers) indicating a cigarette smoke - but not PAH - caused induction of CYP 1A2 in smokers. As a consequence of the degree of PAH exposure the ratio dihydrodiols/phenols depends on the total amount of metabolites excreted. Phenols predominate, equally in smokers and non-smokers after low exposure, while dihydrodiols become more prominent in highly exposed persons (coke plant workers). Both (i) the regiospecific oxidation of PAH and (ii) the ratio of dihydrodiol vs phenol formation may be recognized from the urinary phenanthrene metabolite profile. This pattern mirrors the enzymatic status (balance of the CYP isoforms and epoxide hydrolase) in individuals. Accordingly, more detailed information may be obtained from the urinary metabolite pattern than from 1- hydroxypyrene, commonly used in PAH biomonitoring.

Regio- and stereoselectivity in the metabolism of benzo[c]phenanthrene mediated by genetically engineered V79 Chinese hamster cells expressing rat and human cytochromes P450.

Regio- and stereoselective metabolism mediated by cytochrome P450 (CYP) and metabolite-dependent cytotoxicity of benzo[c]phenanthrene (B[c]Ph) and its trans-3,4-dihydrodiol, the metabolic precursor of the carcinogenic fjord-region B[c]Ph-3,4-dihydrodiol 1,2-epoxides (B[c]PhDE), were investigated with V79 Chinese hamster cells genetically engineered for three rat and six human CYP isoforms. The order of the capabilities of the CYP isoforms to metabolize B[c]Ph was as follows: h1A1>r1A1>r1A2>h1B1>h1A2>r2B1>>h2E1>h2A6>h3A4. Regardless of the species, all individual CYP isoforms preferentially catalyzed the oxidation of B[c]Ph at the 5,6-position (K-region) except human CYP1A1 and human CYP1A2, which oxidized both the 5,6- and the 3,4-position with similar efficiency. While human CYP1A1, rat CYP1A1 and rat CYP1A2 formed almost exclusively the (-)-B[c]Ph-3R,4R-dihydrodiol, human CYP1A2 produced both the (-)-3R,4R- and the (+)-3S,4S-dihydrodiol enantiomers in a ratio of 2:1. Stereoselective activation of B[c]Ph, the (±)-B[c]Ph-3,4-dihydrodiol and its (-)-3R,4R-enantiomer to the fjord-region (-)-anti-B[c]PhDE occurred upon incubation with rat CYP1A1 and rat CYP1A2 as indicated by the formation of two stereoisomeric tetraols, the hydrolysis products of the labile anti-B[c]PhDE. The formation of tetraols in the culture medium was accompanied by a concentration-dependent increase in cytotoxicity indicating that this effect was mediated by the fjord-region (-)-anti-B[c]PhDE formed as reactive intermediate. All human and rat CYP-expressing V79 cell lines investigated did not show any significant capacity to metabolize the (+)-3S,4S-dihydrodiol. The present study indicates that the human CYP isoforms 1A1 and 1B1 have complementary catalytic properties to activate B[c]Ph to its fjord-region B[c]PhDE, whereas other human isoforms play a minor role. Activation of B[c]Ph by human CYP1A1 and 1B1 is less efficient than by rat CYP1A1 or rat CYP1A2, but proceeds with similar stereoselectivity via the (-)-3R,4R-dihydrodiol to the strong carcinogen (-)-anti-B[c]PhDE with (R,S,S,R)-configuration.

Long-term decline of atmospheric and marine pollution by polycyclic aromatic hydrocarbons (PAHS) in Germany.

Concentrations of benzo[a]pyrene and benzo[e]pyrene in the atmosphere as markers for the class of PAHs decreased by about 70% within one decade in clean air as well as in industrially polluted areas of Germany when measured with passive samplers (spruce sprouts, poplar and beech leaves). The same trend has been found for East-Germany during 1991-1995. Mussels (Mytilus edulis) were found to accumulate PAHs from the aquatic environment and exhibited a seasonal periodicity of the PAH concentration. After an initial decline from 1985 to 1990, the PAH concentration remained constant until 1995 in the North Sea area investigated.

Environmental monitoring and banking of marine pollutants by using common mussels.

Since 1985 the common mussel Mytulis edulis has been collected, characterized and stored within the framework of the German Environmental Specimen Bank program. Selected results about the determination of various pollutants such as heavy metals, polycyclic aromatic hydrocarbons and chlorinated hydrocarbons are presented. Particular emphasis is given to the comparison of seasonal variations with respect to different environmental contaminants and their correlations depending on sampling location and time.

DNA single strand breakage, DNA adducts, and sister chromatid exchange in lymphocytes and phenanthrene and pyrene metabolites in urine of coke oven workers.

OBJECTIVES: To investigate the specificity of biological monitoring variables (excretion of phenanthrene and pyrene metabolites in urine) and the usefulness of some biomarkers of effect (alkaline filter elution, 32P postlabelling assay, measurement of sister chromatid exchange) in workers exposed to polycyclic aromatic hydrocarbons (PAHs). METHODS: 29 coke oven workers and a standardised control group were investigated for frequencies of DNA single strand breakage, DNA protein cross links (alkaline filter elution assay), sister chromatid exchange, and DNA adducts (32P postlabelling assay) in lymphocytes. Phenanthrene and pyrene metabolites were measured in 24 hour urine samples. 19 different PAHs (including benzo(a)pyrene, pyrene, and phenanthrene) were measured at the workplace by personal air monitoring. The GSTT1 activity in erythrocytes and lymphocyte subpopulations in blood was also measured. RESULTS: Concentrations of phenanthrene, pyrene, and benzo(a)pyrene in air correlated well with the concentration of total PAHs in air; they could be used for comparisons of different workplaces if the emission compositions were known. The measurement of phenanthrene metabolites in urine proved to be a better biological monitoring variable than the measurement of 1-hydroxypyrene. Significantly more DNA strand breaks in lymphocytes of coke oven workers were found (alkaline filter elution assay); the DNA adduct rate was not significantly increased in workers, but correlated with exposure to PAHs in a semiquantitative manner. The number of sister chromatid exchanges was lower in coke oven workers but this was not significant; thus counting sister chromatid exchanges was not a good variable for biomonitoring of coke oven workers. Also, indications for immunotoxic influences (changes in lymphocyte subpopulations) were found. CONCLUSIONS: The measurement of phenanthrene metabolites in urine seems to be a better biological monitoring variable for exposure to PAHs than measurement of hydroxypyrene. The alkaline filter elution assay proved to be the most sensitive biomarker for genotoxic damage, whereas the postlabelling assay was the only one with some specificity for DNA alterations caused by known compounds.

Determination of urinary metabolites of polycyclic aromatic hydrocarbons (PAH) for the risk assessment of PAH-exposed workers.

A method for the simultaneous determination of urinary phenanthrene, fluoranthene, pyrene, chrysene and benzo[a]pyrene metabolites has been developed for individual risk assessment at polycyclic aromatic hydrocarbon (PAH)-burdened workplaces. The concentration of urinary metabolites as a measure for individual PAH exposure takes account not only of PAH masses resorbed by the respiratory tract but also those incorporated percutaneously. The method allows the determination of 25 different components with a low margin of error; the individual metabolite profiles thereby allow conclusions on the individual characteristics of PAH-oxidizing enzymes (monooxygenases). The coefficients of variation are lower than 10%. After enzymatic treatment of the urine with glucuronidase and arylsulfatase one part of the benzene or toluene extract is treated with diazomethane to convert phenols into methylethers, while another part is used to convert dihydrodiols into phenols. After further purification the metabolites are determined by means of a combination of gas chromatography and mass spectrometry. The PAH exposure of cock plant workers during several consecutive days resulted in fairly constant individual urinary metabolite profiles which, however, exhibited significant inter-individual variability. This held true also for Wistar rats exposed to tar pitch aerosol on 5 days during a period of 10 days. It was also demonstrated that in the case of coke plant workers there is a correlation between inhaled PAH and metabolites excreted. Mass relationships between inhaled PAH and metabolites excreted were found to differ from one individual to another.

Metabolism and excretion of polycyclic aromatic hydrocarbons in rat and in human.

Polycyclic aromatic hydrocarbons have shown to be an important class of environmental and occupational carcinogens. By balancing the carcinogenic potential PAH were found to predominantly contribute to the biological activity of environmental matter such as vehicle exhaust, used motor oil, and hard-coal combustion effluents. Due to the individual ratio of toxifying and detoxifying processes PAH-exposure measurements are not appropriate to be used for risk assessment without any further information on their metabolic fate. Accordingly, metabolite profiles of phenanthrene, pyrene, chrysene, benz(a)anthracene and fluoranthene have been recorded in both tar-pitch exposed Wistar rats and coke plant workers. The results show that metabolite profiles are invariant individual parameters which, however, vary from one individual to another. Significant differences with regard to the ratio of k-region and non-k-region hydroxylation of phenanthrene have been observed in a greater number of coke plant workers. This ratio might be helpful for risk assessment studies since it reflects the various cytochrome P450-dependent monooxygenase isoforms participating in the metabolism of PAH. Studies of this kind can only be carried out with substrates possessing several nonequivalent double bonds (phenanthrene, chrysene) whereas pyrene--commonly used for biomonitoring--does not satisfy this condition. The excretion rate (excretion versus exposure) seems to be an individual parameter.

Cytochrome P450-mediated activation of phenanthrene in genetically engineered V79 Chinese hamster cells.

V79 Chinese hamster cells genetically engineered for rat cytochromes P450 1A1, 1A2, 2B1 and human cytochromes P450 1A1, 1A2, 2A6, 2E1, and 3A4 are being applied in metabolism studies on polycyclic aromatic hydrocarbons. This study presents the results on phenanthrene as the prototypic polycyclic aromatic hydrocarbon possessing a bay region. Phenanthrene is of less importance regarding cytotoxicity and carcinogenicity as compared to e.g. benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene. However, phenanthrene is more readily converted to metabolites which are exreted in higher amounts than those from any other polycyclic aromatic hydrocarbon. Therefore, its metabolites are of diagnostic value in epidemiological and occupational exposure studies. For this reason, it is worthwhile to understand the metabolism of phenanthrene in detail, e.g. allocating metabolites and cytochromes P450s. In accordance to previous observations cytochromes P450 1A1 and 1A2 were the most active forms towards phenanthrene. However, metabolite profiles differed between rat and human homologues of cytochromes P450, in particular for cytochrome P450 1A2. The predominant metabolite formed by rat cytochrome P450 1A2 was the K region trans-9,10-dihydrodiol, whereas human cytochrome P450 1A2 produced similar amounts of the trans-1,2-, trans-3,4- and trans-9,10-dihydrodiol. High amounts of trans-1,2-dihydrodiol, the metabolic precursor of the bay-region dihydrodiol epoxide, were also formed by human cytochrome P450 1A1 compared to its rat homologue. Unexpectedly, human cytochrome P450 2E1 showed a remarkable catalytic activity to metabolize phenanthrene to its trans-9,10-dihydrodiol. Utilizing recombinant CYPs in live V79 cells appears to be a valuable too yielding results important for the evaluation of exposure data and risk assessment for humans.

Urinary metabolite profile of PAH as a potential mirror of the genetic disposition for cancer.

Polycyclic aromatic compounds such as polycyclic aromatic hydrocarbons or aromatic amines presently are considerably underestimated with regard to the formation of environmentally caused cancer diseases. The individual urinary metabolite profile raising from the PAH inhaled is invariant. This holds for tar-pitch aerosol exposed Wistar rats as well as for PAH-exposed workers. Significant individual differences of the urinary metabolite profile can be observed in different individuals. The differences reflect the different individual enzyme equipment. There is an individual correlation between the PAH-masses inhaled and the masses of their metabolites excreted in the urine; e.g. the excretion of phenanthrene varies from 5% to 20% for different coke workers. The PAH metabolite profile analysis appears to be a suitable tool to estimate the individual cancer.risk at PAH-exposed working places since the PAH-induced malign transformation is caused by specific PAH metabolites.

Comparison of total DNA adduct levels induced in mouse tissues and human skin by mainstream and sidestream cigarette smoke condensates.

Cigarette smoke condensates (CSCs) of both mainstream (MS) and sidestream (SS) smoke were used to treat mice topically in equivalent amounts. Human skin maintained in short-term culture was also treated with the condensates. DNA adducts, induced by the CSCs and detected by the nuclease P1 method of 32P-postlabelling, were quantified in a number of murine tissues and in the human skin DNA. In the five mouse tissues studied both MS-CSC and SS-CSC produced characteristic diagonal radioactive zones on TLC, indicative of the formation of multiple DNA adducts. In three tissues (skin, lung and kidney), SS-CSC induced greater total adduct levels than MS-CSC (statistically significant in skin and kidney, p < 0.05). However, greater adduct levels induced by MS-CSC were recorded for heart and bladder DNA (not statistically significant). Similar results to those found in mouse skin were obtained with human skin; SS-CSC induced a approximately 2-fold greater level of DNA adducts than MS-CSC (p < 0.05). Incubation of DNA directly with condensates in vitro demonstrated that DNA adducts could be formed without an exogenous metabolizing system. This direct interaction of condensates with DNA occurred at similar levels for both MS- and SS-CSC, although inclusion of an oxygen radical-generating system enhanced the SS-CSC binding to a greater extent than that of the MS-CSC.

Biomonitoring of polycyclic aromatic hydrocarbons in highly exposed coke plant workers by measurement of urinary phenanthrene and pyrene metabolites (phenols and dihydrodiols).

A filter combination consisting of an impregnated glass fibre and a control filter was used for the collection of air samples in which gaseous and particulate polycyclic aromatic hydrocarbons (PAHs) were determined. To estimate the loss of lower boiling PAHs, d10-phenanthrene was applied as internal standard. A simple, well-producible method for the determination of 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, 1,2-, 3,4- and 9,10-dihydroxydihydrophenanthrene, 1-hydroxypyrene and 1,2-dihydroxy-1,2-dihydropyrene is described. By means of personal air samplers the exposure to PAHs of four coke plant employees working at different locations was measured over 4 days. Simultaneously the 24-h urine was collected and stored frozen until analysed. The main excretion product of pyrene is a 1-hydroxypyrene conjugate, whereas phenanthrene is excreted predominantly as dihydrodiol conjugate. As expected, workers on the battery topside were exposed the most and accordingly excreted by far the highest amounts of PAHs. Up to 34.0 micrograms phenanthrol conjugates (total of all isomeric phenols) and 195.5 micrograms dihydrodiol conjugates (total of all isomeric dihydrodiols) were excreted in the 24-h urine (mean of 4 days). The metabolite profiles of five isomeric phenanthrene phenols and three isomeric dihydrodiols exhibited only small percentage variations within one individual whereas significant interindividual differences were observed. These findings may indicate a genetically determined enzyme pattern responsible for the metabolic conversion of PAHs.

Covalent binding of polycyclic aromatic hydrocarbon components of coal tar to DNA in mouse skin.

Treatment of mouse skin with coal tar is known to initiate tumour formation, with the carcinogenic activity associated mainly with polycyclic aromatic hydrocarbons (PAHs). A sample of pharmaceutical coal tar was analysed by gas chromatography and 19 major PAHs were identified. 32P-postlabelling analysis was used to characterize those PAHs that are responsible for the DNA binding of coal tar and, by inference, its biological activity. PAHs were grouped according to their reported carcinogenic activities and applied as mixtures to mouse skin. Group A contained all of the 19 PAHs, group B seven PAHs for which there is sufficient evidence for carcinogenicity and group C 12 PAHs with only limited or inadequate evidence of carcinogenicity in experimental animals. 32P-Labelled DNA adducts formed by coal tar were resolved on TLC into a pattern of three discrete spots (2, 4 and 6) and four areas of diffuse radioactivity (1, 3, 5 and 7). By comparison of the pattern of adducts formed by coal tar with those formed by the synthetic mixtures it appeared that PAHs in group B formed coal tar-DNA adduct spots 4 and 6, and that adduct spot 2 was formed by PAHs in group C. Attempts to identify those PAHs responsible for the formation of coal tar-DNA adducts 4 and 6 were made by comparing the chromatographic mobilities of 32P-labelled coal tar-derived DNA adducts formed in mouse skin, using TLC and HPLC, with those formed by PAHs in group B. As benzo[ghi]perylene (B[ghi]P), a component of group C, has been demonstrated to exhibit significant DNA binding ability previously, the chromatographic mobility of coal tar-DNA adduct spot 2 was compared to that of the major DNA adducts formed by B[ghi]P in vivo and in vitro. It appeared that coal tar adduct spot 2 was the major adduct formed by B[ghi]P in vitro and that benzo[a]pyrene, benzo[b]fluoranthene, benzo [j]fluoranthene and benzo[k]fluoranthene contributed to the formation of adduct spot 6. None of the PAHs examined appeared to be responsible for the formation of adduct spot 4.

DNA adduct formation in mice following treatment with used engine oil and identification of some of the major adducts by 32P-postlabelling.

Used engine oil from a petrol-powered vehicle was fractionated by column chromatography into seven parts for which the major polycyclic aromatic hydrocarbon (PAH) components were determined by GC. Topical treatment of mice with the fractions and 32P-postlabelling of the skin DNA resulted in the detection of multiple adduct spots on TLC for some, but not all, of the fractions. The majority of the DNA binding capacity of the used engine oil was possessed by the first three fractions, (equivalent to 25, 15 and 14.5%, respectively) of the adduct forming ability of the unfractionated oil. The chromatographic mobilities of the adduct spots induced by these fractions were compared to those produced by unfractionated used engine oil. In addition, mice were also treated topically with reference PAHs, either singly or as mixtures, dissolved in unused oil at the concentrations at which they were present in the used oil. Comparisons were made between the chromatographic mobilities of the adducts formed in mouse skin DNA by synthetic mixtures with those formed by the used oil. From these data, some of the major adducts produced by treatment with used engine oil are suggested to be formed by reactive metabolites of benzo[b]naphtho[1,2-d]thiophene, benzo[c]phenanthrene, benzo[g,h,i]fluoranthene, chrysene, benzo[a]pyrene and benzo[g,h,i]perylene.

Rat liver microsomal ring- and S-oxidation of thiaarenes with central or peripheral thiophene rings.

The metabolism of the following thiaarenes has been investigated using liver microsomes of untreated, phenobarbital-, AroclorR- and 5,6-benzoflavone-pretreated rats: dibenzothiophene, naphtho[2,1-b]thiophene, benzo[b]naphtho[2,1-b]thiophene, benzo[b]naphtho[1,2-d]thiophene, benzo[b]naphtho-[2,3-d] thiophene, phenanthrol[1,2-b]thiophene, phenanthrol[4,5-bcd] thiophene, triphenyleno[1,12-bcd]-thiophene and dinaphtho[2,1-b:1',2'-d]thiophene. Thiaarenes with a central thiophene ring preferentially undergo S-oxidation and are converted into sulfoxides and sulfones, whereas those with a peripheral thiophene ring are oxidized at the carbocyclic skeleton resulting in the formation of phenols, dihydrodiols and triols. Sulfone formation seems to be inducible by phenobarbital but only little or not by 5,6-benzoflavone treatment. In most cases 5,6-benzoflavone and AroclorR treatment enhanced the rates of ring oxidation.

Analysis of the polycyclic aromatic hydrocarbon content of petrol and diesel engine lubricating oils and determination of DNA adducts in topically treated mice by 32P-postlabelling.

Engine lubricating oils are known to accumulate carcinogenic polycyclic aromatic hydrocarbons (PAHs) during engine running. Oils from nine petrol-powered and 11 diesel-powered vehicles, in addition to samples of unused oil, were analysed for PAH content and ability to form DNA adducts when applied topically to mouse skin. The levels of 19 PAHs, determined by GC, were in total, approximately 22 times higher in used oils from petrol engines than in oils from diesel engines. Male Parkes mice were treated with 50 microliters of oil daily for 4 days before they were killed and DNA isolated from skin and lung tissue. DNA samples were analysed by nuclease P1-enhanced 32P-postlabelling. Used oils from both diesel and petrol engines showed several adduct spots on PEI-cellulose plates at total adduct levels of up to 0.57 fmol/microgram DNA [approximately 60 times greater than in experiments with samples of unused oil in which adduct levels (0.01-0.02 fmol adducts/microgram DNA) were close to the limit of detection]. Higher adduct levels were generally formed by petrol engine oils than by diesel engine oils. Lung DNA contained similar total adduct levels to those in skin although the adduct maps were less complex. Total adduct levels correlated with extent of oil use in the engine, the total PAH concentration in oils and with the concentrations of certain individual PAHs present in the oils. An adduct spot that co-eluted with that of the major benzo[a]pyrene-DNA adduct accounted for 9-26% of the total adducts in skin DNA, and approximately 8% of the adducts in lung DNA, of mice treated with petrol engine oils. A major, and as yet unidentified, adduct spot comprised up to 30% of the total adducts in skin DNA, and up to 89% of the total adducts in lung DNA, of these animals.

Dietary influences on urinary excretion of hydroxyphenanthrenes, thioethers and mutagenicity in man.

Our study indicates that large differences in dietary polycyclic aromatic hydrocarbon (PAH) content in humans are not reflected by urinary and faecal excretion of hydroxyphenanthrenes, although significant increases in 3-hydroxybenzo[a]pyrene and 3-hydroxychrysene in faeces were observed after consumption of a diet rich in PAHs. The question therefore arises whether urinary hydroxyphenanthrenes are a reliable marker for exposure to PAHs. As expected, the elevated mutagenicity of the diet rich in PAHs led to increased mutagenic activity in urine. The increased urinary excretion of thioethers after this diet was probably due to its higher thioether content. Therefore, an elevated thioether excretion does not always indicate exposure to electrophilic compounds.