Matrine and Oxymatrine: evaluating the gene mutation potential using in silico tools and the bacterial reverse mutation assay (Ames test).

The quinolizidine alkaloids matrine and its N-oxide oxymatrine occur in plants of the genus Sophora. Recently, matrine was sporadically detected in liquorice products. Morphological similarity of the liquorice plant Glycyrrhiza glabra with Sophora species and resulting confusion during harvesting may explain this contamination, but use of matrine as pesticide has also been reported. The detection of matrine in liquorice products raised concern as some studies suggested a genotoxic activity of matrine and oxymatrine. However, these studies are fraught with uncertainties, putting the reliability and robustness into question. Another issue was that Sophora root extracts were usually tested instead of pure matrine and oxymatrine. The aim of this work was therefore to determine whether matrine and oxymatrine have potential for causing gene mutations. In a first step and to support a weight-of-evidence analysis, in silico predictions were performed to improve the database using expert and statistical systems by VEGA, Leadscope (Instem®), and Nexus (Lhasa Limited). Unfortunately, the confidence levels of the predictions were insufficient to either identify or exclude a mutagenic potential. Thus, in order to obtain reliable results, the bacterial reverse mutation assay (Ames test) was carried out in accordance with OECD Test Guideline 471. The test set included the plate incorporation and the preincubation assay. It was performed with five different bacterial strains in the presence or absence of metabolic activation. Neither matrine nor oxymatrine induced a significant increase in the number of revertants under any of the selected experimental conditions. Overall, it can be concluded that matrine and oxymatrine are unlikely to have a gene mutation potential. Any positive findings with Sophora extracts in the Ames test may be related to other components. Notably, the results also indicated a need to extend the application domain of respective (Q)SAR tools to secondary plant metabolites.

Genotoxicity assessment: opportunities, challenges and perspectives for quantitative evaluations of dose-response data.

Genotoxicity data are mainly interpreted in a qualitative way, which typically results in a binary classification of chemical entities. For more than a decade, there has been a discussion about the need for a paradigm shift in this regard. Here, we review current opportunities, challenges and perspectives for a more quantitative approach to genotoxicity assessment. Currently discussed opportunities mainly include the determination of a reference point (e.g., a benchmark dose) from genetic toxicity dose-response data, followed by calculation of a margin of exposure (MOE) or derivation of a health-based guidance value (HBGV). In addition to new opportunities, major challenges emerge with the quantitative interpretation of genotoxicity data. These are mainly rooted in the limited capability of standard in vivo genotoxicity testing methods to detect different types of genetic damage in multiple target tissues and the unknown quantitative relationships between measurable genotoxic effects and the probability of experiencing an adverse health outcome. In addition, with respect to DNA-reactive mutagens, the question arises whether the widely accepted assumption of a non-threshold dose-response relationship is at all compatible with the derivation of a HBGV. Therefore, at present, any quantitative genotoxicity assessment approach remains to be evaluated case-by-case. The quantitative interpretation of in vivo genotoxicity data for prioritization purposes, e.g., in connection with the MOE approach, could be seen as a promising opportunity for routine application. However, additional research is needed to assess whether it is possible to define a genotoxicity-derived MOE that can be considered indicative of a low level of concern. To further advance quantitative genotoxicity assessment, priority should be given to the development of new experimental methods to provide a deeper mechanistic understanding and a more comprehensive basis for the analysis of dose-response relationships.

Use of transcriptomics in hazard identification and next generation risk assessment: A case study with clothianidin.

Toxicological risk assessment is essential in the evaluation and authorization of different classes of chemical substances. Genotoxicity and mutagenicity testing are of highest priority and rely on established in vitro systems with bacterial and mammalian cells, sometimes followed by in vivo testing using rodent animal models. Transcriptomic approaches have recently also shown their value to determine transcript signatures specific for genotoxicity. Here, we studied how transcriptomic data, in combination with in vitro tests with human cells, can be used for the identification of genotoxic properties of test compounds. To this end, we used liver samples from a 28-day oral toxicity study in rats with the pesticidal active substances imazalil, thiacloprid, and clothianidin, a neonicotinoid-type insecticide with, amongst others, known hepatotoxic properties. Transcriptomic results were bioinformatically evaluated and pointed towards a genotoxic potential of clothianidin. In vitro Comet and γH2AX assays in human HepaRG hepatoma cells, complemented by in silico analyses of mutagenicity, were conducted as follow-up experiments to check if the genotoxicity alert from the transcriptomic study is in line with results from a battery of guideline genotoxicity studies. Our results illustrate the combined use of toxicogenomics, classic toxicological data and new approach methods in risk assessment. By means of a weight-of-evidence decision, we conclude that clothianidin does most likely not pose genotoxic risks to humans.

First onset of treatment of patients with eating disorders and treatment course: Results of data from a German health insurance company.

OBJECTIVE: This study examines, inpatient treatment costs, and typical treatment courses of patients with an eating disorder using secondary data. METHOD: The data were provided by a German health insurance company (data from 4.2 million members from 2004 to 2010; corresponds to a market share of 6% of all statutorily insured persons in Germany). An age and gender matched control group without an eating disorder diagnosis was assessed for comparisons from the same dataset. RESULTS: Two thousand seven hundred and thirty four cases with an eating disorder diagnosis (anorexia nervosa [AN], bulimia nervosa [BN] or combination [ANBN]) were identified. The inpatient costs of treatment were €5471.15 for BN, €9080.26 for AN, €10,809.16 for ANBN and €339.37 for the control group. Interestingly, there are numerous mild episodes of eating disorders that could be successfully treated solely on an outpatient basis with a short treatment duration. CONCLUSION: Our findings suggest that course and severity of eating disorders can vary from mild to very severe. Data from health insurance companies depict rather different disease and treatment courses than studies on primary data derived from treatment institutions.

Alkenylbenzenes in Foods: Aspects Impeding the Evaluation of Adverse Health Effects.

Alkenylbenzenes are naturally occurring secondary plant metabolites, primarily present in different herbs and spices, such as basil or fennel seeds. Thus, alkenylbenzenes, such as safrole, methyleugenol, and estragole, can be found in different foods, whenever these herbs and spices (or extracts thereof) are used for food production. In particular, essential oils or other food products derived from the aforementioned herbs and spices, such as basil-containing pesto or plant food supplements, are often characterized by a high content of alkenylbenzenes. While safrole or methyleugenol are known to be genotoxic and carcinogenic, the toxicological relevance of other alkenylbenzenes (e.g., apiol) regarding human health remains widely unclear. In this review, we will briefly summarize and discuss the current knowledge and the uncertainties impeding a conclusive evaluation of adverse effects to human health possibly resulting from consumption of foods containing alkenylbenzenes, especially focusing on the genotoxic compounds, safrole, methyleugenol, and estragole.

[Providing Rapid Help for Mental Strain at the Workplace: Psychosomatic Consultation in the Workplace]. / Rasche Hilfe bei psychischen Belastungen am Arbeitsplatz: Die Psychosomatische Sprechstunde im Betrieb.

BACKGROUND: Psychosomatic consultation in the workplace (PSIW) is an offer for employees who are under mental and psychosomatic strain. Core elements are early diagnosis and short-term psychotherapy with the aim of improving the care for mentally stressed employees. This article provides a characterization of patients and presents initial data on the effects of short-term psychotherapy. MATERIALS AND METHODS: From 05/2016 to 12/2019, basic data were collected from all employees seeking help. Socio-demographic data, previous treatments, work ability, depression (PHQ-9), anxiety (GAD-7), somatic symptoms (PHQ-15), assessment of psychological state and attitudes towards treatment options were collected by self-report before and (if applicable) after a short-term intervention. RESULTS: A total of 672 employees from 20 companies (49% male) were mostly referred to PSIW by the company physician. Adjustment disorders and depressive disorders each accounted for almost one-third of diagnoses. A quarter of the employees presenting at PSIW were on sick leave at the time of referral. The most frequent recommendations were short-term intervention at PSIW, followed by outpatient psychotherapy. A total of 343 (51%) employees completed the questionnaires, of which 187 (55%) of them received short-term psychotherapy. The symptoms improved significantly across all scales. Satisfaction with PSIW was very high. CONCLUSIONS: PSIW is an innovative care concept in the work context, which is well accepted and effective in various sectors. Close cooperation with company stakeholders such as occupational health physicians is important for a successful outcome.

Early intervention, treatment and rehabilitation of employees with common mental disorders by using psychotherapeutic consultation at work: study protocol of a randomised controlled multicentre trial (friaa project).

BACKGROUND: Common mental disorders are one of the leading causes for sickness absence and early retirement due to reduced health. Furthermore, a treatment gap for common mental disorders has been described worldwide. Within this study, psychotherapeutic consultation at work defined as a tailored, module-based and work-related psychotherapeutic intervention will be applied to improve mental health care. METHODS: This study comprises a randomised controlled multicentre trial with 1:1 allocation to an intervention and control group. In total, 520 employees with common mental disorders shall be recruited from companies being located around five study centres in Germany. Besides care as usual, the intervention group will receive up to 17 sessions of psychotherapy. The first session will include basics diagnostics and medical indication of treatment and the second session will include work-related diagnostics. Then, participants of the intervention group may receive work-related psychotherapeutic consultation for up to ten sessions. Further psychotherapeutic consultation during return to work for up to five sessions will be offered where appropriate. The control group will receive care as usual and the first intervention session of basic diagnostics and medical indication of treatment. After enrolment to the study, participants will be followed up after nine (first follow-up) and fifteen (second follow-up) months. Self-reported days of sickness absence within the last 6 months at the second follow-up will be used as the primary outcome and self-efficacy at the second follow-up as the secondary outcome. Furthermore, a cost-benefit assessment related to costs of common mental disorders for social insurances and companies will be performed. DISCUSSION: Psychotherapeutic consultation at work represents a low threshold care model aiming to overcome treatment gaps for employees with common mental disorders. If successfully implemented and evaluated, it might serve as a role model to the care of employees with common mental disorders and might be adopted in standard care in cooperation with sickness and pension insurances in Germany. TRIAL REGISTRATION: The friaa project was registered at the German Clinical Trial Register (DRKS) at 01.03.2021 (DRKS00023049): https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00023049 .

Parasitism in Gambusia affinis: Fitness Effects in an Incipient Matrotroph.

Gambusia affinis (western mosquitofish) serves as a host for a variety of larval and adult parasites. Gambusia affinis is also an incipient matrotroph, exhibiting adjustments in post-fertilization provisioning to some offspring within a brood using recently acquired resources. Nutrient transfer to embryos is expected to limit the loss of embryo mass during development resulting in larger offspring. Since larger offspring are more likely to survive, maternal contributions are expected to increase fitness. The presence of parasites, particularly intestinal helminths, potentially reduces body condition and resources available for developing offspring, thereby reducing host fitness. The effects of parasitism on the fitness of G. affinis were investigated in the present study. Fish were collected from 3 sites monthly from June 2015 through August 2016. All helminth parasites were collected during necropsy and identified. Brood size and embryo developmental stage were recorded for each female fish. Additionally, 10 ova/embryos of each developmental stage from each female fish collected from May through August 2016 were haphazardly selected and individually weighed. From 429 female mosquitofish, 5,072 helminths were collected. Brood size varied among collection sites and was positively influenced by maternal body condition, the number of daylight hours, water temperature, and the intensity of both plerocercoid and adult Schyzocotyle acheilognathi. However, brood size was negatively related to the intensity of Neoechinorhynchus cylindratus cystacanth and an increasing number of days between collection and dissection. Embryo weight increased with the presence of either Camallanidae or Contracaecum multipapulatum, embryo developmental stage, and relative host density. These results indicate that some parasitic helminth species negatively affect the fitness of G. affinis, while some positively affect fitness, and that effect can vary with intensity.

Variation in Helminth Parasite Component Communities of Gambusia affinis.

In north-central Texas streams, seasonal changes in ambient temperature and rainfall result in dynamic variation in microhabitat structure, affecting the distribution and abundance of hosts. Since the complex life cycles of parasites require the presence and interaction of multiple hosts, the seasonal variations in microhabitats could influence parasite component communities within these ecosystems. Gambusia affinis (western mosquitofish) serves as a host for a variety of larval and adult parasites. Factors that affect helminth component communities in populations of mosquitofish from the Paluxy River were investigated in this study. Gambusia affinis individuals were collected from 3 sites monthly from June 2015 through August 2016. All helminth parasites were collected during necropsy and subsequently identified. From 495 mosquitofish, 5,283 helminths were collected. Component community diversity varied among collection sites and collection dates and was positively influenced by river flow. These results indicate that helminth assemblages infecting mosquitofish in the Paluxy River vary through time and space, as well as in response to environmental variation (i.e., changes in water flow).

(Q)SAR tools for the prediction of mutagenic properties: Are they ready for application in pesticide regulation?

The assessment of human health risks resulting from the presence of metabolites in groundwater and food residues has become an important element in pesticide authorisation. In this context, the evaluation of mutagenicity is of particular interest and a paradigm shift from exposure-triggered testing to in silico-based screening has been recommended in the European Food Safety Authority (EFSA) Guidance on the establishment of the residue definition for dietary risk assessment. In addition, it is proposed to apply in silico predictions when experimental mutagenicity testing is not possible due to a lack of sufficient quantities of the pesticide metabolite. This, combined with animal welfare and economic considerations, has led to a situation where an increasing number of in silico studies are submitted to regulatory authorities. Whilst there is extensive experience with in silico predictions for mutagenicity in the chemical and pharmaceutical industry, their suitability in pesticide regulation is still insufficiently considered. Therefore, we herein discuss critical issues that need to be resolved to successfully implement (Quantitative) Structure-Activity Relationship ((Q)SAR) as an accepted tool in pesticide regulation. For illustration purposes, the results of a pilot study are included. The presented study highlights a need for further improvement regarding the predictivity and applicability domain of (Q)SAR systems for pesticides and their metabolites, but also raises other questions such as model selection, establishment of acceptance criteria, harmonised approaches to the combination of model outputs into overall conclusions, adequate reporting and data sharing. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels.

Methyleugenol is a rodent hepatocarcinogen occurring in many herbs and spices as well as essential oils used for flavoring. Following metabolic activation by cytochromes P450 (CYPs) and sulfotransferases (SULTs), methyleugenol can form DNA adducts. Previously, we showed that DNA adduct formation by methyleugenol in mouse liver is dependent on SULT1A1 expression and that methyleugenol DNA adducts are abundant in human liver specimens. In humans, SULT1A1 activity is affected by genetic polymorphisms, including single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). Here we investigated the relationship between individual methyleugenol DNA adduct levels and SULT1A1 in human liver samples. Using isotope-dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry, we quantified methyleugenol DNA adducts in 121 human surgical liver samples. Frequent CNVs, including deletions (f = 3.3%) and duplications (f = 36.4%) of SULT1A1, were identified using qPCR and TaqMan assays in the donors' genomic DNA. SULT1A1 mRNA and protein levels were quantified using microarray data and Western blot analysis, respectively. Methyleugenol DNA adducts were detected in all 121 liver samples studied. Their levels varied 122-fold between individuals and were significantly correlated to both mRNA and protein levels of SULT1A1 (r s = 0.43, and r s = 0.44, respectively). Univariate and multivariate statistical analysis identified significant associations of SULT1A1 CNVs with mRNA (p = 1.7 × 10-06) and protein (p = 4.4 × 10- 10) levels as well as methyleugenol DNA adduct levels (p = 0.003). These data establish the importance of SULT1A1 genotype for hepatic methyleugenol DNA adducts in humans, and they confirm a strong impact of SULT1A1 CNVs on SULT1A1 hepatic phenotype.

Microhabitat Selection and Eyefluke Infection Levels in the Western Mosquitofish (Gambusia affinis).

Several trematode species infect the eyes of fish as second intermediate hosts. In most cases the definitive host is a piscivorous bird. Studies of a few species have shown an increase in transmission due to decreased visual acuity of the fish host. However, this may vary depending on trematode microhabitat choice within the eye. Some trematode species are found in the lens, some are found in the vitreous humor, and others have been reported from the retina. Here we report 3 genera of eyeflukes in 3 locations of the eye in the intermediate fish host, Gambusia affinis . Clinostomum metacercariae were found attached to the outer sclera within the eye orbit, and Diplostomum metacercariae were found in the lens. Posthodiplostomum metacercariae were confirmed by histology to reside between the choroid and pigmented retina. Posthodiplostomum metacercariae were found in both eyes of all 20 fish examined and in high intensities (up to 27 metacercariae per eye). High trematode intensities between the choroid and pigmented retina found in this study may disrupt vision in this fish host. Our study is the first to document the microhabitat of all 3 trematode metacercariae within the eye of G. affinis .

Simultaneous detection of multiple DNA adducts in human lung samples by isotope-dilution UPLC-MS/MS.

Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using liquid chromatography connected to tandem mass spectrometry (LC-MS/MS). However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from 16 ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by ultra performance liquid chromatography (UPLC)-MS/MS. Methods for the enrichment of the analytes from DNA hydrolysates and chromatographic separation preceding mass spectrometric analysis were optimized, and the resultant technique was used for the simultaneous analysis of the 16 DNA adducts in human lung biopsy specimens. Eleven adducts (formed by benzo[a]pyrene, 1-methylpyrene, 4-aminobiphenyl, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 1-methoxy-3-indolylmethylglucosinolate, 5-hydroxymethylfurfural, and malondialdehyde) were not detected in any tissue sample (limits of detection: 0.02-7.1 adducts/10(8) nucleosides). 3,N(4)-etheno-2'-deoxycytidine and 1,N(6)-etheno-2'-deoxyadenosine, formed from 2,3-epoxyaldehydes of endogenous lipid peroxidation products, were present in all subjects (16.9-115.3 and 27.2-179/10(8) nucleosides, respectively). The same was true for N(2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine, the major adduct of methyleugenol (1.7-23.7/10(8) nucleosides). A minor adduct of methyleugenol and two adducts of furfuryl alcohol were detected in several pulmonary specimens. Taken together, we developed a targeted approach for the simultaneous mass spectrometric analyses of 16 DNA adducts, which can be easily extended by adducts formed from other mutagens. The method allowed one to detect adducts of furfuryl alcohol and methyleugenol in samples of human lung.

Formation of hepatic DNA adducts by methyleugenol in mouse models: drastic decrease by Sult1a1 knockout and strong increase by transgenic human SULT1A1/2.

Methyleugenol--a natural constituent of herbs and spices--is hepatocarcinogenic in rodent models. It can form DNA adducts after side-chain hydroxylation and sulfation. We previously demonstrated that human sulfotransferases (SULTs) 1A1 and 1A2 as well as mouse Sult1a1, expressed in Salmonella target strains, are able to activate 1'-hydroxymethyleugenol (1'-OH-ME) and 3'-hydroxymethylisoeugenol (3'-OH-MIE) to mutagens. Now we investigated the role of these enzymes in the formation of hepatic DNA adducts by methyleugenol in the mouse in vivo. We used FVB/N mice [wild-type (wt)] and genetically modified strains in this background: Sult1a1 knockout (ko), transgenic for human SULT1A1/2 (tg) and the combination of both modifications (ko-tg). Methyleugenol (50mg/kg body mass) formed 23, 735, 3770 and 4500 N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adducts per 10(8) 2'-deoxyribonucleosides (dN) in ko, wt, ko-tg and tg mice, respectively. The corresponding values for an equimolar dose of 1'-OH-ME were 12, 1490, 12 400 and 13 300 per 10(8) dN. Similar relative levels were observed for the minor adduct, N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine. Thus, the adduct formation by both compounds was nearly completely dependent on the presence of SULT1A enzymes, with human SULT1A1/2 producing stronger effects than mouse Sult1a1. Moreover, a dose of 0.05 mg/kg methyleugenol (one-fourth of the estimated average daily exposure of humans) was sufficient to form detectable adducts in humanized (ko-tg) mice. Although 3'-OH-MIE was equally mutagenic to 1'-OH-ME in Salmonella strains expressing human SULT1A1 or 1A2, it only formed 0.14% of hepatic adducts in ko-tg mice compared with an equimolar dose of 1'-OH-ME, suggesting an important role of detoxifying pathways for this isomer in vivo.

Abundance of DNA adducts of methyleugenol, a rodent hepatocarcinogen, in human liver samples.

Methyleugenol is a genotoxic carcinogen in mice and rats, the liver being the primary target tissue. Methyleugenol occurs in fennel and many herbs and spices. Furthermore, methyleugenol-containing plant extracts and chemically prepared methyleugenol are used as flavoring agents. We analyzed surgical human liver samples from 30 subjects for the presence of DNA adducts originating from methyleugenol using isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Twenty-nine samples unambiguously contained the N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adduct. A second adduct, N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine, was also found in most samples, but at much lower levels, in agreement with the results from experimental models. The maximal and median levels of both adducts combined were 37 and 13 per 10(8) nucleosides (corresponding to 4700 and 1700, respectively, adducts per diploid genome). This is the first demonstration of DNA adducts formed by a xenobiotic in human liver using UPLC-MS/MS, the most reliable method available. It has been estimated for diverse rat and mouse hepatocarcinogens that 50-5500 adducts per 10(8) nucleosides are present after repeated treatment at the TD50 (daily dose that halves the probability to stay tumor-free in long-term studies). We conclude that the exposure to methyleugenol leads to substantial levels of hepatic DNA adducts and, therefore, may pose a significant carcinogenic risk.

Optimized enzymatic hydrolysis of DNA for LC-MS/MS analyses of adducts of 1-methoxy-3-indolylmethyl glucosinolate and methyleugenol.

Mass spectrometric analyses of DNA adducts usually require enzymatic digestion of the DNA to nucleosides. The digestive enzymes used in our laboratory included a calf spleen phosphodiesterase, whose marketing was stopped recently. Using DNA adducted with bioactivated methyleugenol and 1-methoxy-3-indolylmethyl glucosinolate-each forming dA and dG adducts-we demonstrate that replacement of calf spleen phosphodiesterase (Merck) with bovine spleen phosphodiesterase (Sigma-Aldrich) leads to unchanged results. Enzyme levels used for DNA digestion are extremely variable in different studies. Therefore, we sequentially varied the level of each of the three enzymes used. All dose (enzyme)-response (adduct level) curves involved a long plateau starting below the enzyme levels employed previously. Thus, we could reduce the amounts of micrococcal nuclease, phosphodiesterase, and alkaline phosphatase for quantitative DNA digestion by factors of 4, 2, and 333, respectively, compared to our previous protocols. Moreover, we observed significant phosphatase activity of both phosphodiesterase preparations used, which may affect the recovery of adducts with methods requiring digestion to 2'-deoxynucleoside-3'-monophosphates (e.g., (32)P-postlabeling). In addition, the phosphodiesterase from Sigma-Aldrich, but not that from Merck, deaminated dA. This was irrelevant for the dA adducts studied, involving bonding at N(6), but might complicate the analysis of other dA adducts.

Inter-rater reliability of the ICPC-2 in a German general practice setting.

QUESTIONS: Three- and four-digit International Classification of Diseases (ICD-10) is not a reliable classification system in primary care. The reliability of the International Classification of Primary Care (ICPC-2) as an alternative coding system has not yet been investigated in a German general practice setting. METHODS: Cross-sectional data were collected during a one year period in a general practice setting. PARTICIPANTS: A total of 8,877 patients were randomly selected. MAIN OUTCOME MEASURES: The first of the reasons for encounter was taken into account on new and chronic managed problems. The ICPC-2 coding of each case was performed by two raters to investigate the inter-rater agreement. The degree of agreement between the raters was assessed by using Cohen's kappa (κ ≥ 0.61 meaning high or satisfactory and κ ≤ 0.6 (incl. ≤ 0.000) meaning low or unsatisfactory). RESULTS: The reliability was good to excellent at the chapter level, at the component level the reliability was moderate though good in the components 1-symptoms and 7-diseases. At single code level the agreement was only fair to moderate in both chapters and components. One third to half of the used codes showed good inter-rater agreement. CONCLUSION: The ICPC-2 is an adequate and feasible instrument for routine use in general practice. The fair to moderate reliability on the single code level should be considered when designing studies and interpreting data that are based on the ICPC-2.

Metabolism of methyleugenol in liver microsomes and primary hepatocytes: pattern of metabolites, cytotoxicity, and DNA-adduct formation.

Methyleugenol (1) is a constituent of many foods, in particular of herbal spices, and is used as flavoring agent in foodstuffs and as fragrance in cosmetics. 1 has been found to be carcinogenic in rodents, its metabolite, 1-hydroxymethyleugenol (2) acting as proximate DNA-binding carcinogen. We incubated 1 with liver microsomes of rat, bovine, and human origin. We found 2, 3-hydroxymethylisoeugenol (3), and 6-hydroxymethyleugenol (4) as major metabolites, and 1-oxomethyleugenol (5), 3-oxomethylisoeugenol (6), eugenol (9), chavibetol (11), and (RS)-2,3-dihydroxy-2,3-dihydromethyleugenol (7) as minor metabolites. Methyleugenol-2,3-epoxide (8), probably the precursor of 7, could not be detected. Incubations with synthetic metabolites were applied in order to uncover metabolic pathways. Incubations with primary rat hepatocytes revealed mainly nonconjugated 2 and conjugated 4, and minor amounts of partly conjugated 7 and conjugated 9 + 11. The "reactive metabolites" 3, 5, 6, and 8 were not detectable, possibly due to rapid reaction with cellular macromolecules. The highest cytotoxicity (resazurin reduction assay and lactate dehydrogenase leakage assay) was observed for the main metabolite 2 and its secondary metabolite 5 with EC(50) values of 50 and 10 µM, respectively. Deoxyadenosine or deoxyguanosine adducts were formed by incubating 1 or metabolites with rat hepatocytes. The rank order of adduct formation was 2 > 1 > 3 > 6, whereas 4, 5, and 8 were inactive. In conclusion, we present a virtually complete pattern of microsomal (rat, bovine, and human) and hepatocellular (rat) metabolites of 1 suggesting the formation of several reactive metabolites possibly involved in carcinogenicity, organ toxicity, and immune reactions.

Primary care research - trade-off between representativeness and response rate of GP teachers for undergraduates.

PRINCIPLES: Low response rates are common in primary care research. Our study examines the representativeness of respondents in a survey among general practitioners (GPs). One special aim was to evaluate the representativeness of the subgroup of GP teachers for undergraduates (GPTUs) and to investigate the option of a panel of GPTUs. METHODS: The representativeness of the respondents was assessed by the use of pooled public data to compare the respondents and all GPs in the German federal state of Saxony on the basis of socio-demographic and subject-specific characteristics. The representativeness of the GPTUs was examined in the same way. For the analysis, two-sided t-tests and Chi2 tests were used. RESULTS: The total response rate was low (32.87%). The respondents were not a representative sample; in particular, they were more highly qualified than the mean. However, the response rate among the special group of university-associated GP teachers for undergraduates was significantly higher than among other general practitioners. Because of this, the creation of a panel of these GPTUs for further primary care research was investigated. Unfortunately, analysis of this group showed that GPTUs were not a representative sample as they tended to be younger and more highly qualified. CONCLUSIONS: In general it is possible to create a panel of GPTUs to obtain higher response rates, but investigation of the panel's representativeness is definitely required. If the panel is not representative another option is the creation of a stratified sample according to the target population.

Identification of human and murine sulfotransferases able to activate hydroxylated metabolites of methyleugenol to mutagens in Salmonella typhimurium and detection of associated DNA adducts using UPLC-MS/MS methods.

Methyleugenol, a secondary metabolite present in many herbal spices, is carcinogenic in various tissues of mice and rats but negative in standard in vitro mutagenicity tests. Several observations indicate that hydroxylation followed by sulfation is an important activation pathway in the carcinogenicity and DNA adduct formation by methyleugenol and other alkenylbenzenes in animal models. However, sulfation is not taken into account in standard in vitro tests. Therefore, we have studied whether expression of murine or human sulfotransferases (SULTs) in the target strain, Salmonella typhimurium TA100, leads to the activation of hydroxylated metabolites of methyleugenol [(+)-1'-hydroxymethyleugenol, (-)-1'-hydroxymethyleugenol and (E)-3'-hydroxymethylisoeugenol]. Human SULT1A1 (a form expressed at high levels in many tissues) and SULT1C2 (expressed primarily in foetal tissues) activated all three compounds even at very low substrate concentrations. At higher concentrations, activation was also observed with human SULT1A2 and SULT1E1. Murine Sult1a1 required higher substrate concentrations than its human orthologue. Other SULT forms (human 1A3, 1C1, 1C3, 2A1 and 2B1b as well as murine 1d1) did not activate any methyleugenol metabolites studied. Furthermore, we developed isotope-dilution mass-spectrometric methods for the sensitive and specific detection of DNA adducts formed by methyleugenol metabolites. All three hydroxylated metabolites formed the same DNA adducts in S. typhimurium TA100-hSULT1A1: high levels of N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine and modest levels of N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine. Adduct levels correlated with the mutagenic effects induced. No adducts were formed by the test compounds in the SULT-deficient standard strain TA100. In conclusion, several methyleugenol metabolites are activated to DNA-reactive mutagens in S. typhimurium upon incorporation of appropriate sulfation capacity. We have identified human and murine SULT forms able to catalyse this activation. Methods were developed that may be utilised to analyse DNA samples from human tissues specifically for the possible presence of methyleugenol adducts.