Hum. Genet. 49, 147--158 (1979) © by Springer-Verlag 1979

Chromosome Distribution Studies After Inorganic Lead Exposure L. Verschaeve*, M. Driesen, M. Kirsch-Volders, L. Hens, and C. Susanne Laboratorium voor Antropogenetica, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium

Summary. We studied the chromosome distribution in persons professionally exposed to inorganic lead. The degree of lead exposure was evaluated by biochemical measurements and cytogenetic analysis. The chromosome distribution was analyzed from trypsin banded karyotypes; in particular we studied centromere distances (A2) and centromere-metaphase-center distances (d 2) which were obtained by computer-aided mathematical transformation of the individual metaphase coordinates. Higher concentrations of blood lead and urine d-ALA and a statistically significant increase in aneuploidy, hypoploidy, and type-B chromosome aberrations revealed appreciable exposure although none of the subjects showed signs of excessive lead absorption. However study of the chromosome distribution showed no major differences with that of the controls indicating that lead acts preferentially (directly or indirectly) on the chromosomes rather than on the spindle apparatus. A dissociation of the acrocentric chromosomes was observed in the lead group when compared with the controls. This is thought to reflect a secondary action of lead on the nucleolar organiser regions. Introduction Since many studies have demonstrated a nonrandom distribution of chromosomes in normal human mitotic metaphases (Hoo and Cramer, 1971; Warburton et al., 1973; Hens et al., 1975; Kirsch-Volders et al., 1977), we may assume that chemical or physical factors that influence the mitotic processes directly or indirectly (for example interaction with the spindle apparatus) will change the normal distribution pattern. This was observed in persons professionally exposed to phenyl-mercury-acetate (Verschaeve et al., 1978) and was at least partially * To whom offprint requests should be sent

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L. Verschaeve et al.

e x p l a i n e d t h r o u g h t h e k n o w n r e a c t i v i t y o f m e r c u r y c o m p o u n d s w i t h sulfh y d r y l g r o u p s in t h e s p i n d l e a p p a r a t u s . T h e s t r o n g a f f i n i t y f o r m e r c a p t a n e s is h o w e v e r n o t t h e sole p r o p e r t y o f m e r c u r y c o m p o u n d s b u t is a l s o e n c o u n t e r e d , t h o u g h less p r o n o u n c e d , f o r o t h e r h e a v y m e t a l s like l e a d a n d c a d m i u m (Vallee a n d U l m e r , 1972). B a s e d o n this s i m i l a r i t y in b i o c h e m i c a l b e h a v i o r we t h u s m a y e x p e c t a n a l t e r e d c h r o m o s o m e d i s t r i b u t i o n in p e r s o n s p r o f e s s i o n a l l y e x p o s e d to lead. W e r e p o r t h e r e t h e results o f a c h r o m o s o m e d i s t r i b u t i o n s t u d y p e r f o r m e d o n p e r s o n s e x p o s e d to i n o r g a n i c l e a d a n d o n age r e l a t e d c o n t r o l s . T h e s e results m a y give us s o m e n e w i n s i g h t a b o u t the m e c h a n i s m o f a c t i o n o f l e a d c o m p o u n d s at t h e c e l l u l a r a n d c h r o m o s o m a l levels.

Materials and Methods Blood samples were obtained from nine workers occupationally exposed to inorganic lead (mean age 37.8 years). All nine men showed no signs of intoxication or obvious excessive lead absorption, although in the past some of them had been periodically transferred to a lead-free work environment or even temporarily discharged because of clinical and preclinical signs of intoxication. At the time of sampling, which occurred during routine medical supervision, all subjects were considered 'normalized', that is as being in a 'normal' state of health. No exposure to environmental factors other than lead could be traced. They furthermore had no history of significant disease including genetic disorders. Blood samples from the controls were obtained from an organization for blood transfusion. The controls (12 men, mean age 40.5 years) had previously served as controls in a chromosome distribution study after exposure to phenylmercury-acetate (Verschaeve et al., 1978). The exposure level to lead was estimated from biochemical and standard cytogenetic analyses. Lead concentrations in whole blood were determined by the method of Roosels and Bossiroy (in preparation). The hemoglobin content in the blood and ~-ALA concentration in the urine were also determined in the lead-exposed subjects. Chromosome analysis was performed by standard procedures on lymphocytes from 48 h cultures. For structural chromosome aberrations and chromosome count an average of 100 cells per subject in both the exposed group and the controls were analyzed after Giemsa staining. For the study of chromosome distribution, a total of 100 metaphase plates were karyotyped in the control group (eight subjects with 10 metaphases and four with 5 metaphases), and a total of 89 metaphase plates in the lead exposed group (eight subjects with 10 metaphases and one with 9 metaphases. Karyotypes were made after trypsin banding by the method of Klinger (1972). The identification of the chromosome pairs after banding allowed us to study the chromosome distribution in both exposed and control groups. The methodology used in this study is given in detail in other papers (Hens et al., 1975; Hens, 1976; Galperin-Lema~tre et al., 1977; etc.). Briefly, the Coordinates of all chromosomes in each metaphase plate were recorded and a circular transformation was then performed according to Barton et al. (1965) to obtain the 'generalized square distance A2' (between the centromeres of two chromosomes and the 'generalized square distance d z' (between the centromere of a chromosome and the center of gravity of the metaphase). These values, obtained with the aid of a CDC 6400 computer, are dimensionless, and the procedure allows a direct comparison between the different metaphases independent from their form and photographic magnification. Within both investigated populations, d 2 and A 2 for a given chromosome combination were transformed into percentage frequencies and arranged in histogram classes of equal intervals. Comparison with a reference distribution allowed us to decide if a given chromosome combination showed a specific distribution pattern. The reference distribution was obtained by adding all d 2 ( o r A 2 values) for all possible chromosome combinations. But to make the population of numbers in the reference distribution and that of the particular histogram under study independent of each other, the absolute class frequencies of the reference distribution were diminished with the absolute class frequencies of the particular histogram.

Chromosome Distribution Studies After Inorganic Lead Exposure

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2'2 tests allow us to decide within a group (A2 or d 2 of one chromosome combination with corresponding reference distribution) or between the two investigated groups (A2 or d: in the control group with the same A2 or d 2 in the exposed group) whether the compared histograms belong to the same population of numbers. For A2 generalized distances, supplementary data were obtained by studying the 'association tendency' as defined by Galperin et al. (1977). Here 2z tests were performed for only two histogram classes: the first included the absolute values corresponding to the first column of the previously described histograms containing the lowest A2 values; the second class contained all the rest of the absolute values. Again, both cell populations were compared. Special attention was also paid to combinations of the D- and G-group chromosomes since they are known to associate preferentially (Cook, 1972; Galperin-Lema~tre et al., 1977; and others) and a specific action on those acrocentrics would not be surprising according to our previous results for mercury (Verschaeve et al., 1978). Finally, cumulative frequencies of A2 distances were also studied for both populations for the following combinations: within and between acrocentrics and within and between homologous non acrocentrics.

Results Cytogenetic and Biochemical Analyses B i o c h e m i c a l an d cytogenetic analyses were p e r f o r m e d to evaluate the degree o f exposure. L e a d c o n c e n t r a t i o n s in the b l o o d ranged between 50 an d 8 4 y % in the e x p o s ed p o p u l a t i o n (mean: 65.6) a n d between 10 and 25~% in the c o n t r o l p o p u l a t i o n (mean: 16.25). Thus the b l o o d lead c o n c e n t r a t i o n s o f all exposed

Table 1. Results of the cytogenetic analysis Lead-exposed group

Controls

Statistics

a) Chromosome count a % aneuploidy

(range) (mean)

15.00--33.62 26.65

9.57--36.14 19.67

P< 0.005

% hypoploidy

(range) (mean)

10.00--29.31 22.66

5.32--33.73 15.50

P< 0.005

(range) (mean)

1.12-- 6.50 3.98

1.20-- 8.64 4.17

N.S.

(range) (mean)

3.41--20.79 9.96

0.00-- 8.11 3.24

P< 0.005

(range) (mean)

0.00-- 8.60 0.89

0.00-- 1.56 0.98

N.S.

% hyperploidy

b) Chromosome aberrations b % type-B cells % type-C cells

High aneuploidy values are thought to be due to the technique used for chromosome analysis (Verschaeve et al., 1978) b Type-B cells: cells with chromatid-type aberrations (gap, isogap, single breaks) Type-C ceils: ceils with chromosome-type aberrations (isobreak, dicentrics, etc.) Nomenclature as in Buckton et al., 1962

L. Verschaeve et al.

150 Table 2.

d2

values for the control group and the lead exposed group

Control group

Lead group

Chromosome

d2

Probability~

Chromosome

d2

Probability

22 15 14 13 21 16 1 17 19 9 18

1.584 1.676 1.698 1.775 1.816 1.824 1.824 1.880 1.924 1.971 1.986

P< 0.005 0.005 < P < 0.01 P< 0.005 0.01 < P < 0.025 N.S. 0.01< P< 0.025 N.S. N.S. N.S. N.S. N.S.

1.578 1.676 1.714 1.795 1.809 1.861 1.905 1.960 1.976 1.992 2.000

P< 0.005 0.01 < P < 0.025 P< 0.005 N.S. N.S. N.S. N.S. N.S. N.S. N.S.

Mean distance 2 10 12 11 5 8 6 7 3 20 4 X XY Y

2.000

22 21 15 1 14 17 11 12 16 10 Mean distance 13

2.004

0.025 < P< 0.05

19 7 5 4 20 9 3 X XY Y 2 18 8 6

2.034 2.038 2.056 2.061 2.073 2.077 2.087 2.102 2.138 2.173 2.179 2.253 2.262 2.476

N.S. N.S. N.S. N.S. N.S. N.S. 0.025< P< 0.01 N.S. N.S. N.S. N.S. N.S. N.S. 0.01

Chromosome distribution studies after inorganic lead exposure.

Hum. Genet. 49, 147--158 (1979) © by Springer-Verlag 1979 Chromosome Distribution Studies After Inorganic Lead Exposure L. Verschaeve*, M. Driesen, M...
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