Appendix D - Dossier (PDF) - Tera

date: 20–JUL–2005
5. Toxicity Substance ID: 71–43–2
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Endpoint: other: human genetic polymorphism and benzene
metabolism
Method: SUBJECTS AND METHODS
Benzene exposure (personal diffusive sampling with
subsequent analysis by GC–FID) was assessed over the course
of a work week (Monday morning to Friday evening; approx.
110 hr) for 40 subjects (20 men, 20 women, age 27–46 yr)
living and working in central Copenhagen. Ten of the
subjects worked partly outdoors (e.g. policemen), the
reminder in an office environment. They answered a
questionnaire that included questions of possible sources of
benzene exposure, smoking habits, type of work etc.
Appendix D: Benzene SIDS Dossier
A sample of venous blood (heparinised) and a 24 hr urine
sample were collected from all subjects on day 1 of the
study.
ANALYSIS OF 8–OXOdG AND COMET ASSAY
Washed lymphocytes were isolated by centrifugation, lysed
and DNA isolated using published methods (Wellejus and Loft
(2002) FASEB J 16:195). The concentration of
7–hydro–8–oxo–2’–deoxyguanosine (8–oxodG) in lymphocyte DNA
and in urine was then determined using HPLC with
electrochemical detection.
A separate sample of lymphocytes was processed for
single–cell electrophoresis (comet assay; Moller et al.
(2001) FASEB J 15: 1181) after enzymic digestion
(fapyguanine glycosylase, endonuclease). The cells were
scored visually for damage (0 = no damage; 4 = maximum
damage) and summed (giving a total score between 0 and 400).
BIOMARKERS OF BENZENE EXPOSURE IN URINE
Trans,trans muconic acid (t,t–MA) and S–phenylmercapturic
acid (S–PMA) were quantified using GC–MS.
DETERMINATION OF GSTT1, GSTM1, GSTP1 AND NQO1 GENOTYPES
DNA was isolated from lymphocytes (phenol extraction) and
used as a template for polymerase chain reaction (2 cycles)
prior to agarose gel electrophoresis. After staining with
ethidium bromide and visualisation with UV light, the
genotypes GSTT1 (Pemble et al. (1994) Biochem J 300, 271),
GSTM1 (Zhong et al. (1991) Carcinogenesis 12, 1533), GSTP1
(Harries et al. (1997) Carcinogenesis 18, 641) and NQO
(Wiencke et al. (1997) Cancer Epidem Biomarker Prev. 6, 87)
were determined by PCR based assays, following published
methods.
STATISTICAL METHODS
Demographic data, urinary 8–oxodG and exposure data were
analysed according to gender using Mann–Whitney tests.
Differences in t,t–MA and S–PMA excretion according to NQO,
GSTP1, GSTM1 and GSTT1 genotype status were analysed using
multifactorial ANOVA. Analyses for 8–oxodG in lymphocytes
and results from the comet assay were evaluated using
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