Biological Monitoring and Genetic Polymorphism - ACS Symposium

Chapter 17

Biological Monitoring and Genetic Polymorphism

Downloaded by UNIV MASSACHUSETTS AMHERST on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0654.ch017

Toshihiro Kawamoto and Yasushi Kodama Department of Environmental Health, University of Occupational and Environmental Health, P.O. Orio, Yahata nishi ku, Kitakyushu 807, Japan

This study focused on clarifying the effects of the genetic polymorphism of ALDH2 (low K aldehyde dehydrogenase) on toluene metabolism. The study subjects were 92 male toluene workers and 234 non-exposed men. The genotype of ALDH2 was classified into the homozygous genotype of normal ALDH2 (NN), the homozygous genotype of an inactive one (DD) and the heterozygous genotype of normal and inactive ones (ND) by means of PCR-RFLP. The personal exposure levels to toluene were monitored using diffusion type samplers and the measurement of urinary hippuric acid (HA) and creatinine concentrations. In the toluene workers, positive correlations between the personal exposure to toluene and the urinary HA levels were observed in the NN, ND and DD groups in comparison to the non-exposed groups. The urinary HA levels of the DD and ND groups were significantly lower than that of the NN group. m

Toluene is one of the most widely used organic solvents in the world. The exposure levels of workers to toluene are estimated by their urinary hippuric acid (HA) level, the main metabolite of toluene (7). In developed countries the measurement of urinary HA level is one of the most important items during the medical check up of toluene workers. Especially in Japan, employers must have urinary HA of toluene workers measured every 6 month by law. Approximately 80 % of the absorbed toluene is first converted to benzyl alcohol by the microsomal mixed function oxidase system, then oxidized to benzoic acid by the alcohol dehydrogenase/aldehyde dehydrogenase system, and finally conjugated with glycine to form HA (2). There are three main isozymes of aldehyde dehydrogenase (ALDH, EC: 1.2.1.3), namely, ALDH1 (high K ALDH, of cytosolic origin), ALDH2 (low K ALDH, of mitochondrial origin) and ALDH3. It is believed that ALDH2 oxidizes most of the acetaldehyde derived from the metabolism of alcohol. Genetic polymorphism in ALDH2 activity is seen in Japanese and other Mongoloids, as well as in American m

m

© 1997 American Chemical Society

In Environmental Biomonitoring; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

17.

KAWAMOTO & KODAMA

Biological Monitoring & Genetic Polymorphism 191


Indians. About half of the Japanese lack ALDH2 activity and experience facial flushing, tachycardia, nausea and hypotension owing to their low capability to metabolize acetaldehyde when they drink alcohol (3). The sequences of cDNA and genomic DNA of ALDH2 have been clarified (4). The catalytic deficiency is caused by a structural point mutation at the amino acid position 487 of the polypeptide subunit of the ALDH2 gene. At this position, a substitution of Glu to Lys resulting from the transition of G(C) to A(T) at DNA levels occurs (5). It is suspected that the metabolism of toluene in ALDH2-deficient workers is different from that in normal ALDH2 workers. Subjects and Methods The study subjects were 92 male workers (ages 18-66 years) who handled toluene in a printing factory, an electric parts factory, and a painting workplace and 234 male university students and workers who were not exposed to toluene. Peripheral venous blood samples (7 ml) were obtained from all these individuals. Genomic DNA was isolated by the standard phenol/chloroform extraction method using a DNA extractor (Applied Biosystems Inc, model-340A) after complete digestion with 100 μg/ml of proteinase Κ (Wako Pure Chemical). The genotypes of ALDH2 were determined by artificial restriction fragment length polymorphism using MhoII (6). The details were already reported by Kawamoto et al. (7). The breathing-zone personal exposure levels to toluene were monitored a diffusion type sampler (Dupont Protek, G-AA). The workers attached the personal samplers to their chests or collars from the beginning to the end of their work shifts. The solvent collected on the sampler was desorbed in carbon disulfide (1.5 ml) overnight and quantified by gas chromatography with an frame ionization detector. Urine was collected in the afternoon from 3:00-5:00 PM. All samples were stored at -20°C until analysis. The urinary hippuric acid and creatinine concentration were determined by a high performance liquid chromatography (8). The measured HA concentration was corrected for creatinine. The differences in the slopes and intercepts of the regression lines were examined by the analysis of Armitage and Berry (9, 70). Results Fig. 1 shows the relationship between the personal exposure to toluene and the urinary HA by the genotypes of ALDH2. Positive correlations were observed in the NN, ND and DD groups. The 95% ranges of the slopes and intercepts for the regression lines, as well as the correlation coefficients, are shown in Table L The slopes of the regression lines decreased from NN, to ND to DD in this order. The intercepts for the regression lines were not significantly different for ALDH2 genotypes. Discussion Benzoic acid, which is a precursor of hippuric acid, is contained in cinnamon, strawberry, etc. (77) and some beverages (72). However, the dietary variation of urinary hippuric acid is too small in comparison with the increase of urinary hippuric acid by toluene exposure (above 1 g/g creatinine) (10, 13). Kawamoto et al. (14) reported that the distribution of urinary hippuric acid measurements in the DD group were significantly different from those in both the NN and ND groups from a study of 890 urine samples from 253 toluene workers. Therefore, it can be said that the genetic polymorphism of ALDH2 should be taken into consideration when the biological monitoring of toluene is done during the medical check-up.


In Environmental Biomonitoring; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997. 3

Personal exposure (mg/m )

3


Fig. 1 Relationship between personal exposure to toluene and urinary hippuric acid by ALDH2 genotypes (The dotted curves mean the 95% confidence range for the mean hippuric acid.)

3




55 28 9

116 98 17

3

Exposed Nonexposed

Number of subjects

85.8 94.4 74.2

G.M.

!)

3.30 3.77 3.00

G.S.D.

2)

Personal exposure

J

6.7- ] * * η (6.3-7.2) ** (5.2 - 6.1) 5.7-J (3.5 - 5.4) 4.5

a (x 0.001)

1}

2)

r 177 (131 - 222) 0.92 209 (164 - 255) 0.92 257 (168 - 346) 0.90

β (x 0.001)

Correction for Creatinine

a and β are parameters of calculated regression lines of y = α χ + β, where χ is the personal exposure concentration to toluene (in mg/m ) and y is hippuric acid concentration in urine (g/g creat. for values corrected for creatinine concentration). **P

Biological Monitoring and Genetic Polymorphism - ACS Symposium

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