cytogenetic effects in lymphocytes of stainless steel welders.

S. No cytogenetic effects in lymphocytes of stainless steel welders. Scand j work environ health 9 (1983) 259-264. In 24 manual metal arc stainless steel welders (means: exposure time 19 years, 100 electrodesld, air chromium level 81 pglm3, urinary chromium 47 pmol/mol creatinine) and 24 matched referents, lymphocytes in peripheral blood were analyzed for cytogenetic effects. No statistically significant differences were observed as to frequency of cells with breaks and fragments (1.5 % for the welders, 1.9 % for the referents); gaps and isogaps (1.8 vs 2.0 %); interchanges, dicentrics, rings and markers (0.8 vs 0.5 %); total number of cells with structural aberrations (4.1 vs 4.4 %); hyperdiploidy (0.3 vs 0.2 %); or total number of cells with aberrations (4.4 vs 4.6 %). Neither were there any differences in the frequencies of micronuclei (7.8 vs 7.9 per mille) or sister chromatid exchanges (11 vs 12 per cell) in lymphocytes of peripheral blood.

and mammalian cells (8,15,21), as well as positive in the mammalian spot test (14). Chromium has been suspected to be a causative agent (8,15). The mutagenic effect seems to be diminished or eliminated in the case of welding fumes (8), as well as in the case of chromium (7,19,28) in the presence of rat liver microsome fraction or human erythrocyte lysate (28). Microsomal preparation from rat lung was however a poor inactivator of chromium mutagenicity (28). Recently a retrospective cohort study of mortality suggested an association between lung cancer and stainless steel welding (31). In addition a slight excess of lung cancer deaths was noted among welders with mixed exposure (29). Similarly a proportionate mortality study reported that welders and flamecutters had an excess of lung and urinary bladder cancer (22).
In the present communication we report a study of the cytogenetic effects in vivo in lymphocytes from peripheral blood of stainless steel manual metal arc welders and matched referents.

Subjects
Welders. Twenty-four male manual metal arc welders from six industries in different parts of Sweden were studied. They were selected because of long and intense welding on stainless steel (table 1). The electrodes and materials used during the last five to ten years contained about 20 (range 12-27) r/o chromium, 10 (range 0-23) C/o nickel (electrodes up to 60 C/o in a few cases and for shorter time periods), and mostly about 3 (range 0-10) 70 molybdenium. In an interview the average number of electrodes used per day during the last decade was estimated by 19 of the welders to be about 100 (table 1). This figure was "checked" by the counting of electrodes used by 17 welders during a workday in which air levels were measured (see the Methods section). The average number used then was 98 (range . The diameter of the electrodes was between 3.25 and 5.0 mm, in a few cases 2.5 mm. The corresponding current used was 100 to 250 A, in a few cases 80 A. Some of the welders under study used welding shields, and others used respiratory protective devices when working in narrow spaces, while others used no such equipment. All industries had general ventilation; some also had local exhaust systems. Referents. For each welder a referent was matched according to sex, age (within 5 years), smoking (table l), socioeconomic class, living area, and drug consumption. Both subjects in one pair used a betablocker as antihypertensive treatment. A rough match on alcohol consumption was also made. None of the referents had any occupational (or other) experience with the handling of stainless steel (or other known mutagenic/carcinogenic agents). Their occupations were electrician (one), dairy worker (two), municipal worker (four), office clerk (two), policeman (one), postman (one), railway clerk (four), sawmill worker (six), and butcher (three).

Methods
Medical examinations. The welders and referents were interviewed for medical and occupational history. Special attention was paid to viral infections and exposure to ionizing radiation, heavy metals, and organic solvents. Routine blood analyses were performed. In addition renal function was studied in some detail. In no subject was there evidence of significant disease (except for the two cases of treated hypertension already mentioned).
Chromium in air. The exposure to chromium in air was determined during one workday (Monday). Representative air samples were collected for each welder in 2-h periods on four cellulose acetate filters (Millipore, pore diameter 0.8 pm) by use of a portable pump (MSA or Casella; 2 llmin). Chromium was analyzed by flameless atomic absorption spectrometry (Perkin-Elmer 403) after the filter was treated with water (soluble chromium VI) and aqua regalis (total chromium). The tirneweighted average exposure for one workday was calculated (table 1).
Chromium in urine. Urine samples were collected during the workday. In this study the samples used were obtained immediately after the end of work (table 1). The analysis of chromium in urine was made with a direct flameless atomic absorption spectrometric method. The detection limit was 0.008 pmol/l. The method error was 0.02 pmolll in the range 0-0.2 pmolll and 0.1 pmolll in the range 0.6-2 pmolll. The accuracy of the method was confirmed by good results in repeated interlaboratory checks.
Cytogenetic studies. Lymphocytes were obtained from venous blood samples taken in the afternoon at the same time from each pair. The samples were shipped by air to the laboratory. Latency time before the start of cultivation was about 16 h. Each cytogenetic parameter was analyzed by one observer, who was unaware of the exposure data. Chromosome aberrations were assessed by a routine microculture method described earlier (9). Cultivation time was 72 h. For each individual 100 metaphases were analyzed. The aberrations were scored according to Evans et a1 (5).
Micronuclei were analyzed in lymphocytes cultured for 96 h in the presence of phytohemagglutinin (4,30). After centrifugation the cells were suspended in an equal amont of medium, smeared on a slide, stained according to May-Grunewald-Giemsa (without previous hypotonic treatment or fixation), and scored for micronuclei. For each of 40 subjects (includes 19 pairs) 1,000 lymphocytes were screened.
The sister chromatid exchange frequency was studied in 9 welders and 12 referents (eight pairs included). The sister chromatid exchanges were analyzed in phytohemagglutinin-stimulated peripheral blood lymphocytes cultured for 72 h and stained with the Giemsa technique (27). 5-Bromodeoxyuridine was present during the entire culture period at a final concentration of 0.1 mmol/ml of medium. in cells with 46 chromosomes were analyzed.
Statistical analysis. Wilcoxon matchedpairs signed-ranks test (for the welderreferent pairs) and the analysis of covariance (with regard to exposure, age and smoking) were used. All the p-values reported are two-tailed.
The frequencies of different types of chromosome aberrations did not differ between the welders and referents (when analyzed statistically in pairs or by covariance). This statement applies to the frequency of cells with breaks and fragments When analyzed by covariance, there was a statistically significantly (p < 0.007) higher frequency of cells with interchanges, dicentrics, ring chromosomes, and marker chromosomes (asymmetrical or incomplete symmetrical chromosometype aberrations) in smokers as compared to nonsmokers plus exsmokers. However the frequency of cells with other types of aberrations did not differ, neither did the total number of cells with aberrations. No difference in micronuclei was observed between the welders and referents (means 7.8 vs 7.9 per mille) (fig 21, and there was no effect of smoking. There was no difference in the frequency of sister chromatid exchanges between the welders and referents (1 vs 12 per cell) (fig  3), and no difference between the smokers and nonsmokers.

Discussion
Intense and long-time exposure to manual metal arc welding fumes from stainless steel did not give rise to cytogenetic effects in blood lymphocytes. No cytogenetic damage in welders was found, in comparison to referents, in a similar study from Finland (10). Only one of the cytogenetic parameters studied showed an effect of smoking. This is not quite in accordance with earlier studies and could be due to the small number of subjects studied or to t h e fact that the subjects were not heavy smokers (less than one pack of cigarettes per day).
The lack of cytogenetic effects between the welders and referents in our study might b e explained in several ways. Ingredients in manual metal arc welding fumes from stainless steel may not be mutagenic in man. The workroom exposure to mutagenic agent(s) may be too low, or there may be an insufficient distribution of mutagen(s) to t h e lymphocytes, eg, d u e to retention in the lungs (2,13,16) or to metabolic inactivation of mutagen(s) or t o a mopping u p of chromium by the red blood cells (6, 17). I n addition, mutagenic agent(s) may reach t h e lymphocytes but for some reason do not generate cytogenetic damage in this type of cell, or cytogenetically damaged lymphocytes may be efficiently eliminated from the blood stream. The lack of cytogenetic damage in lymphocytes could, however, still b e in accordance with a mutagenic/ carcinogenic effect in the lungs or other organs. Of course cytogenetic studies of this kind do not exclude t h e possibility of mutagenic effect, eg, point mutations.