Problems in the biological monitoring of chromium(VI) exposed individuals

The usefulness of currently available techniques for the biological monitoring of chromium(VI) exposed individuals is reviewed. Chromium levels in body fluids, such as urine and blood plasma, are reliable markers of exposure to chromium in oxidation states (VI) and (III) and provide a measure of the internalized dose of chromium. These markers are sufficiently sensitive to be useful in most occupational settings encountered today. In contrast, the majority of cytogenetic surveillance studies among chromium platers, ferrochromium workers and stainless steel welders using the manual metal arc (MMA) method have yielded negative or inconclusive results. As a marker for genotoxicity, the number of sister chromatid exchanges in blood lymphocytes proved to be relatively insensitive towards exposure to chromium(VI). There were however significant increases in rare chromosome aberrations among MMA stainless steel welders, although the reported levels of all aberrations combined were similar to those observed among control groups of many other studies. The relative lack of success of cytogenetic surveillance studies using blood lymphocytes is surprising in view of the strong genotoxicity of chromium(VI). A possible explanation comes from recent studies which showed that the differences in chromium lymphocyte levels between exposed and controls were disproportionately small. Another factor which complicates attempts to correlate genotoxic effects in lymphocytes with the processes giving rise to cancers of the respiratory system is the toxicokinetics of inhaled chromium(VI). Only small fractions of the total inhaled dose are distributed in the body while the bulk of chromium(VI) deposited in the lungs remains there for very long periods of time. The vast majority of lymphocytes will therefore come into contact with chromium(VI) not while travelling through the supporting tissues of the lungs but during their migration through the blood. There they take up chromium(VI) that has leached from the lungs. Blood lymphocytes therefore seem to be inappropriate for the monitoring of the biologically effective dose, and of early biological effects arising from exposure to chromium(VI). Thus there is an urgent need to develop techniques which would allow the non-invasive monitoring of internalized doses of chromium in the lung.     

Problems in the biological monitoring of chromium(VI) exposed individuals

The usefulness of currently available techniques for the biological monitoring of chromium(VI) exposed individuals is reviewed. Chromium levels in body fluids, such as urine and blood plasma, are reliable markers of exposure to chromium in oxidation states (VI) and (III) and provide a measure of the internalized dose of chromium. These markers are sufficiently sensitive to be useful in most occupational settings encountered today. In contrast, the majority of cytogenetic surveillance studies among chromium platers, ferrochromium workers and stainless steel welders using the manual metal arc (MMA) method have yielded negative or inconclusive results. As a marker for genotoxicity, the number of sister chromatid exchanges in blood lymphocytes proved to be relatively insensitive towards exposure to chromium(VI). There were however significant increases in rare chromosome aberrations among MMA stainless steel welders, although the reported levels of all aberrations combined were similar to those observed among control groups of many other studies. The relative lack of success of cytogenetic surveillance studies using blood lymphocytes is surprising in view of the strong genotoxicity of chromium(VI). A possible explanation comes from recent studies which showed that the differences in chromium lymphocyte levels between exposed and controls were disproportionately small. Another factor which complicates attempts to correlate genotoxic effects in lymphocytes with the processes giving rise to cancers of the respiratory system is the toxicokinetics of inhaled chromium(VI). Only small fractions of the total inhaled dose are distributed in the body while the bulk of chromium(VI) deposited in the lungs remains there for very long periods of time. The vast majority of lymphocytes will therefore come into contact with chromium(VI) not while travelling through the supporting tissues of the lungs but during their migration through the blood. There they take up chromium(VI) that has leached from the lungs. Blood lymphocytes therefore seem to be inappropriate for the monitoring of the biologically effective dose, and of early biological effects arising from exposure to chromium(VI). Thus there is an urgent need to develop techniques which would allow the non-invasive monitoring of internalized doses of chromium in the lung.     

Mutagenicity of fume particles from metal arc welding on stainless steel in the Salmonella/microsome test.

Mutagenic activity of fume particles produced by metal arc welding on stainless steel (ss) is demonstrated by using the Salmonella/microsome mutagenicity test described by Ames et al., with strain TA100 (base-pair substitution) and TA98 (frame-shift reversion). Results of a representative but limited selection of processes and materials show that mutagenic activity is a function of process and process parameters. Welding on stainless steel produces particles that are mutagenic, whereas welding on mild steel (ms) produces particles that are not. Manual metal arc (MMA) welding on stainless steel produces particles of higher mutagenic activity than does metal inert gas (MIG) welding, and fume particles produced by MIG welding under short-arc transfer. Further studies of welding fumes (both particles and gases) must be performed to determine process parameters of significance for the mutagenic activity.     

Evidence for the presence of mutagenic compounds other than chromium in particles from mild steel welding

A modified Salmonella/microsome liquid culture assay was used to investigate the mutagenicity of the particulate fraction from mild steel welding. Previous reports have implicated compounds of chromium VI as the mutagenic and toxic agents in welding fumes, since only the particles from welding on stainless steel, which contains 15-25% chromium, were mutagenic, whereas particles from welding on mild steel, which contain less than 0.1% chromium, were not mutagenic or toxic. In this investigation, mild steel particles were shown to contain direct-acting and promutagenic compounds that induced frameshift mutations. The mutagenic agents, which were insoluble in sodium phosphate buffer, did not include chromium VI or organic compounds. Further, the expression of mutation appears to require a cell-particle interaction for the release of the mutagenic species from the particles.     

Sister-chromatid exchanges in human lymphocytes after a non-S-phase incubation period to allow excision DNA repair-in vitro exposure to N-acetoxy-2-acetylaminofluorene and ethylene oxide

Sister-chromatid exchanges (SCE) were analyzed in human peripheral blood lymphocytes at the baseline level, after induction of DNA damage by N-acetoxy-2-acetylaminofluorene (NA-AAF) and ethylene oxide (EO), and after a subsequent 18-h DNA-repair incubation period. There was a significant difference between the baseline SCE frequencies as compared to those after 1 h of NA-AAF or EO treatment. There was no significant difference between the SCE frequencies after 1 h of NA-AAF treatment and those after 18 h of DNA-repair incubation, suggesting that only a low level of NA-AAF damage to DNA had been removed. However, there was a significant difference between the SCE frequencies after 1 h of EO treatment and those after 18 h of DNA-repair incubation, indicating that a significant level of EO induced DNA lesions had been repaired. Thus, it seems likely that the EO induced DNA damage is more easily recognized, and hence more rapidly repaired than the NA-AAF induced damage. The reason for this may be the different chemical nature of the DNA lesions induced, which, in turn, leads to different kinetics of DNA repair.     

Quantification of unscheduled DNA synthesis in mononuclear leukocytes of the horse

This study compares the relationship between N-acetoxy-2-acetylaminofluorene (NA-AAF) and u.v. induced unscheduled DNA synthesis (UDS) and their respective relationships to age and blood pressure in horse mononuclear leukocytes with earlier, similar investigations on human leukocytes. U.v. induced UDS was found to proceed more rapidly than NA-AAF induced UDS. A pronounced lag period associated with the rapid demand for 3H-dThd into DNA after u.v. damage was observed. NA-AAF induced UDS correlated significantly with NA-AAF binding, age and the blood pressure of male horses. UDS values, induced by either method, were about half the level calculated for human leukocytes.     

Correlation of the sister chromatid exchange level with chromosome aberrations induced by chemical mutagens in vivo

The data on the dose dependencies of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations during exposure of mouse bone marrow cells in vivo to 5 alkylating substances are provided. The efficacy of SCE induction was found to be higher than that of chromosomal aberrations. It was established that SCE induced by chemical mutagens in vivo and in vitro are more sensitive and stable tests than chromosomal aberrations.     

Exposure of welders and other metal workers to ELF magnetic fields

This study assessed exposure to extremely low frequency (ELF) magnetic fields of welders and other metal workers and compared exposure from different welding processes. Exposure to ELF magnetic fields was measured for 50 workers selected from a nationwide cohort of metal workers and 15 nonrandomly selected full-time welders in a shipyard. The measurements were carried out with personal exposure meters during 3 days of work for the metal workers and 1 day of work for the shipyard welders. To record a large dynamic range of ELF magnetic field values, the measurements were carried out with “high/low” pairs of personal exposure meters. Additional measurements of static magnetic fields at fixed positions close to welding installations were done with a Hall-effect fluxmeter. The total time of measurement was 1273 hours. The metal workers reported welding activity for 5.8% of the time, and the median of the work-period mean exposure to ELF magnetic fields was 0.18 μT. DC metal inert or active gas welding (MIG/MAG) was used 80% of the time for welding, and AC manual metal arc welding (MMA) was used 10% of the time. The shipyard welders reported welding activity for 56% of the time, and the median and maximum of the workday mean exposure to ELF magnetic fields was 4.70 and 27.5 μT, respectively. For full-shift welders the average workday mean was 21.2 μT for MMA welders and 2.3 μT for MIG/MAG welders. The average exposure during the effective time of welding was estimated to be 65 μT for the MMA welding process and 7 μT for the MIG/MAG welding process. The time of exposure above 1 μT was found to be a useful measure of the effective time of welding. Large differences in exposure to ELF magnetic fields were found between different groups of welders, depending on the welding process and effective time of welding. MMA (AC) welding caused roughly 10 times higher exposure to ELF magnetic fields compared with MIG/MAG (DC) welding. The measurements of static fields suggest that the combined exposure to static and ELF fields of MIG/MAG (DC) welders and the exposure to ELF fields of MMA (AC) welders are roughly of the same level. Bioelectromagnetics 18:470–477, 1997. © 1997 Wiley-Liss, Inc.     

Skin cytogenetic assay for the detection of clastogens-carcinogens topically administered to mice.

A method for assessing the effect of clastogens on mouse skin epidermal cells was devised and applied. Toxic and mutagenic responses in epidermal cells were tested using two known mutagens and carcinogens, urethane (URE) and 7,12-dimethylbenz[a]anthracene (DMBA). Cell generation time, sister-chromatid exchanges (SCE) and chromosomal aberrations (CA) after topical and intraperitoneal (i.p.) treatment were measured in epidermal and bone marrow cells. After topical administration both tissues responded similarly, whereas after i.p. treatment skin cells were less responsive than bone marrow cells. However, the results indicate the validity of this new cytogenetic approach for the assessment of the genotoxicity of compounds applied directly to skin.     

Cytogenetic study of persons occupationally exposed to ethylene oxide.

Ten persons occupationally exposed to ethylene oxide (EO), used in the sterilization of medical instruments, were studied at a hospital. The estimated concentration to which they were exposed was 60-69 ppm, TWA. Peripheral blood samples from 10 workers and 10 controls of the same age and sex were taken to determine the frequency of sister-chromatid exchanges (SCE) and chromosomal aberrations (CA). The mean frequencies of SCE/cell (X = S) were 13.27 for the exposed workers and 6.05 for controls. Chromosome aberration frequencies in exposed individuals were significantly increased compared with controls. A significant relationship between the frequencies of SCE and CA and EO exposure was demonstrated. Blood chemistry parameters such as urea, creatinine, uric acid, lactic dehydrogenase, glutamic oxaloacetic and pyruvic transaminases, luteinizing gonadotropin and follicle stimulating gonadotropin and thyrotropin were also measured and found to be within the normal range.     

Cytogenetic study of persons occupationally exposed to ethylene oxide

Ten persons occupationally exposed to ethylene oxide (EO), used in the sterilization of medical instruments, were studied at a hospital. The estimated concentration to which they were exposed was 60–69 ppm, TWA. Peripheral blood samples from 10 workers and 10 controls of the same age and sex were taken to determine the frequency of sister-chromatid exchanges (SCE) and chromosomal aberrations (CA). The mean frequencies of SCE/cell (X = S) were 13.27 for the exposed workers and 6.05 for controls. Chromosome aberration frequencies in exposed individuals were significantly increased compared with controls. A significant relationship between the frequencies of SCE and CA and EO exposure was demonstrated. Blood chemistry parameters such as urea, creatinine, uric acid, lactic dehydrogenase, glutamic oxaloacetic and pyruvic transaminases, luteinizing gonadotropin and follicle stimulating gonadotropin and thyrotropin were also measured and found to be within the normal range.     

Influence of Neurospora endonuclease on trenimon-induced structural chromosomal aberrations and sister-chromatid exchanges in human peripheral lymphocytes and Chinese hamster ovary cells

Human peripheral lymphocytes and Chinese hamster ovary cells were treated in the G1 phase of the cell cycle with the trifunctional alkylating agent trenimon (TRN) and post-treated with a single-strand specific endonuclease from Neurospora crassa (NE). TRN induces chromosomal aberrations of the chromatid type (CA) and sister-chromatid exchanges (SCE). NE post-treatment leads to an elevation of the frequencies of CA but not of SCEs. This indicates that TRN induced CA are the result of DNA double-strand breaks and that the SCEs originate from other types of lesions, most probably base damage.     

Sister-chromatid exchanges and chromosomal aberrations in a population exposed to pesticides

Sister-chromatid exchanges (SCE) and chromosomal aberrations were studied in a population of floriculturists occupationally exposed to organophosphorus, carbamate and organochlorine pesticides. Blood samples from 36 individuals from a community of 154 persons of asiatic origin were obtained. Among the group sampled, 21 individuals exhibited at least one symptom of chronic intoxication. SCE analysis was performed in 14 symptomatic and 13 asymptomatic persons. The asymptomatic group showed a SCE frequency of 5.47 +/- 1.03 and the symptomatic group a frequency of 6.45 +/- 1.19. Comparison between both groups with the Mann-Whitney 'U' test revealed a significant difference (p 0.0409). Case-control analysis of 9 pairs matched by sex and age also showed significant differences between both groups (p 0.0104). In contrast, the frequencies of chromosomal aberrations were not correlated with intoxication symptomatology, though a significant increment of exchange-type aberrations in relation to a group of non floriculturists was observed in the population studied.     

A cytogenetic study of men environmentally and occupationally exposed to airborne pollutants.

The level of sister-chromatid exchanges (SCE), high-frequency cells (HFC), chromosomal aberrations (CA) as well as the proliferation rate index (PRI) were measured in peripheral blood lymphocytes from three groups of volunteers. The environmentally exposed donors were residents from the vicinity of a coke factory; the occupationally exposed persons were cokery workers, while rural region inhabitants served as a control group. Compared with the control group, statistically significant increases of SCE and HFC, as well as decreased cell kinetics (PRI) were observed for both occupationally and environmentally exposed groups. The effect was especially pronounced when only smokers were taken into account. A statistically significant increase of CA was observed in the environmentally exposed group when CA including gaps (CA + G) were evaluated. The proportion of HFC was found to be the most sensitive method to detect genetic effects on the tested human population. This study demonstrates the usefulness of all 4 biomarkers (SCE, HFC, CA and PRI) in monitoring populations exposed to ambient pollution and clearly indicates effects from residential as well as occupational exposure to industrial air pollutants.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

Evaluation of the mutagenic potential of the hair dye N-methyl-amino-2-nitro-4-N',N'-bis-(2-hydroxyethyl)-aminobenzene in a battery consisting of Drosophila and mammalian cytogenetic assays

The compound N-methyl-amino-2-nitro-4-N', N'-bis(2-hydroxyethyl)-aminobenzene was tested for mutagenic activity in the sex-linked recessive lethal test with Drosophila melanogaster, the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) with Chinese hamster ovary (CHO) cells in vitro, and the micronucleus test with mouse bone-marrow cells in vivo. Consistently negative results were obtained with the 3 tests. The SCE tests gave positive results with prolonged treatments. It is concluded that reliable decisions about mutagenic activity cannot be based on the induction, in vitro, of SCEs alone.     

A cytogenetic study of papaya workers exposed to ethylene dibromide

Ethylene dibromide (EDB) has been shown to be carcinogenic in animal studies and mutagenic in vitro. One cytogenetic study of workers exposed to low levels of EDB for short durations was negative. To test whether exposure to low levels of EDB over long periods caused cytogenetic changes, we have assessed the frequencies of sister-chromatid exchanges (SCE) and chromosomal aberrations (CA) in the peripheral blood lymphocytes of 60 men occupationally exposed to EDB. These men worked in papaya-packing plants where EDB was used to fumigate the fruit after harvest to kill fruit-fly larvae. 42 other men who worked at a nearby sugar mill served as controls. The average duration of exposure of the papaya workers was 5 years. 82 full shift personal breathing-zone air samples indicated that the papaya workers were exposed to a geometric mean of 88 ppb of EDB, as an 8-h time weighted average (TWA). Peaks up to 262 ppb were measured. The proposed OSHA 8-h TWA for EDB is 100 ppb, while NIOSH recommends 45 ppb. No differences in SCE levels were found between exposed and nonexposed workers. No differences were found in the total CA frequency between exposed and nonexposed workers. SCE levels were significantly increased in men who smoked cigarettes (p = 0.0001) and in men who smoked marijuana (p = 0.01). CA levels showed a significant increasing trend with age (p = 0.03).     

Role of homologous recombination in the alpha-particle-induced bystander effect for sister chromatid exchanges and chromosomal aberrations

The bystander effect for sister chromatid exchanges (SCEs) and chromosomal aberrations was examined in hamster cell lines deficient in either DNA-PKcs (V3 cells, deficient in nonhomologous end joining, NHEJ) or RAD51C (irs3 cells, deficient in homologous recombination, HR). Cells synchronized in G0/G1 phase were irradiated with very low fluences of alpha particles such that < 1% of the nuclei were traversed by an alpha particle. Wild-type cells showed a prominent bystander response for SCE induction; an even greater effect was observed in V3 cells. On the other hand, no significant induction of SCE was observed in the irs3 RAD51C-deficient bystander cells irradiated at various stages in the cell cycle. Whereas a marked bystander effect for chromosomal aberrations occurred in V3 cells, the induction of chromosomal aberrations in irs3 bystander cells was minimal and similar to that of wild-type cells. Based on these findings, we hypothesize that HR is essential for the induction of SCE in bystander cells; however, HR is unable to repair the DNA damage induced in NHEJ-deficient bystander cells that leads to either SCE or chromosomal aberrations.     

Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes

The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.