Effect of NOx on the somatic chromosomes of goldsmiths.

The genotoxic effect of NOx was investigated on somatic human chromosomes obtained from lymphocytes of 45 goldsmiths exposed to 1770.5 mg/m3 NOx in ambient air at normal temperature and pressure and compared to an equal number of matched controls breathing air containing 50 microgram/m3 NOx. Short-term lymphocyte cultures were set up from blood collected from both exposed and control individuals by venipuncture in heparinized sterile syringes. Mitotic index (MI), chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and satellite associations (SAs) were analyzed. All the parameters showed a significant increase (p<0.01 and p<0.05) in the exposed individuals as compared to the controls: MI (9.57 vs. 5.01), CAs (3. 48 vs. 0.711), SCEs (10.56 vs. 7.02), and SAs (25.97 vs. 12.84), respectively. Occurrence of DG-type SAs (one D-group chromosome and one G-group chromosome) was highest and 3 D-type (three D-group chromosomes) lowest. NOx was thus found to be genotoxic for humans.

aberrations, genetic hazards, genotoxicity, mitotic index, NO., satellite association, sister chromatid xchng. Environ Heakh Perpect 106: 643-647 (1998). [Online 4 September 1998] hbnp://ebpnetl.niebs.nibgov.docs/d 9,98i106p643-647yadavlabstrahbml NOX (a mixture of NO2 and NO gas) is a red brown toxic gas. When inhaled through the respiratory tract and absorbed through the skin, it causes various skin diseases and respiratory problems. Based on in vitro studies, Beckett et al. (1) postulated that the environmental concentration of HNO2 is formed within the respiratory system predominantly by hydrogen abstraction, with subsequent conversion of HNO2 at physiologic pH, to H+ and NO2. Victorin (X) proposed that HNO2 formed in this way may contribute to the bronchoconstricting effects of NO2 seen in normal subjects and asthmatics. Victorin (2) also observed eye irritation just before, during, and immediately after exposure of asthmatic patients to NO.. Eye irritation was reported to be significantly higher in individuals with HNO2 exposure than in controls. Moreover NO2 levels as low as 0.5 ppm increase susceptibility to bacterial infection of lungs (4. In India, laborers are rarely aware of the potential damage that can accumulate in their genomes due to pollutants in workplaces. NO. gas, evolved from aqua regia used by goldsmiths, is such an example. When gold or silver is added to aqua regia (a mixture of HNO3 and HCR), copper and other metal impurities present in these metals are reduced with the evolution of brown fumes of NO. gas. This prompted us to screen goldsmiths for genetic damage.

Materials and Methods
This study included 45 goldsmiths exposed to NOX and 45 controls matched with respect to age, sex, smoking habits, drinking habits, and social status. Through spectrophotomety the average concentration of NOX in the workplace was found to be 1770.05 mg/m3 compared to 50 pg/m3 at normal temperature and pressure.
We conducted an epidemiological survey using a proforma especially designed for this purpose. After obtaining consent, we performed subsequent medical examinations of subjects suffering from asthma.
Lymphocytes were harvested after 48 hr for determination of mitotic index (MI), chromosomal aberrations (CAs), and satellite associations (SAs). Slides were air dried and stained with 4% Giemsa. As many as 100 good metaphases (well-spread metaphases showing 2N-46 and proper morphology of chromosomes) per individual were screened for CAs.
To determine sister chromatid exchanges (SCEs), we added 5-bromodeoxyuridine (10 pg/ml/culture; Sigma) 24 hr after preparing cultures. We used the method of Perry and Wolff (4) with the following modifications: 1) the cells on slides were stained directly in excess solution of Hoechst 33258 (50 pg/ml in H20; Sigma); 2) the slides were exposed to long-wave UV light of about 320-400 mm intensity; 3) the slides were rinsed with water and incubated for 15-30 min in 2X SSC at 65°C; and 4) the slides were rinsed with water and stained with 2-3% Giemsa in phosphate buffer (pH 6.8). For calculating the frequency of SCEs per cell, 25 (5) were applied: satellite ends of the associating chromosomes had to be directed towards each other with their longitudinal axes meeting between their short arms, and the distance between the centromeres of associated chromosomes could not exceed the total length of one G chromosome, its satellite excluded. The samples were analyzed blindly by two observers to remove possible laboratory scoring bias. For statistical analysis of results, the Student's t-test was applied.

Results
The epidemiological survey showed that 6 individuals out of the sample of 45 goldsmiths were asthmatic. As many as 30 persons also complained of irritation in the eyes.
The data obtained during this study are presented in Tables 1-8. It is evident from Tables 1 and 2 that mean MI in exposed workers (9.57) was significantly higher (p<0.01) than in matched controls (5.01). It was highest in workers with an exposure period of 0-5 years. Thereafter, the MI showed a gradual decline.
We found chromosomal aberrations to be elevated in the group exposed to NOX.
Frequency of SCEs (Table 5) in goldsmiths was significantly higher (10.56; p<0.01) than in controls (7.02). Both among exposed workers and controls, individuals who drink alcohol showed higher SCE frequency (10.30) than controls (7.31). Similarly exposed tobacco smokers showed a higher frequency of SCEs (10.31) than control smokers (7.37). The highest frequency of SCEs (11.02) was observed in an exposed worker who was both a drinker and a smoker (see Table 6). SCEs showed an elevation in frequencies with increase in the duration of exposure ( Table 7). Frequencies of SAs in both exposed and control groups are shown in Table 8. The exposed group showed more than a twofold increase in the frequency of SAs (25.97) compared to controls (12.84). DG association (one D-group chromosome and one G-group chromosome) was found to be highest (7.428%), while 3D association (three D-group chromosomes) showed the lowest frequency (1%).

Discussion
In the present study, 30 out of 45 goldsmiths complained of eye irritation. These 30 persons include 6 asthmatic workers.
There was a significant increase in total CAs in the exposed group as compared to the controls. Chromatid gaps were not taken into account because their significance in cytological monitoring of populations is still a matter of discussion (6). The total chromatid aberrations were more than the total chromosome aberrations in both goldsmiths and controls. A similar elevation in CAs was reported earlier in human populations exposed to polycyclic aromatic hydrocarbons  (PAHs), nitrosamines, carbon monoxide, and sulfur dioxide (7). A significant elevation in the frequency of CAs in peripheral blood lymphocytes among persons belonging to a given group may indicate an increased cancer risk (8,9). Au et al. (10) and Santos-Mello et al. (11) reported that CAs are initial events in carcinogenesis and constitute an early warning signal for development ofcancer. Lu et al. (12 treated mice and rats twice intragastrically with nitrite in doses of 1.7-47 mg/kg body weight and rabbits were given the same dose in drinking water for 3 months; CAs in bone marrow were induced in all three species. In this study, we also found a high frequency of dicentrics. Dicentrics are typical and are normally used as a dosimeter for ionizing irradiation. The present situation, however, reveals that nitric oxides are one of the few NOxs that may confound biological radiation dosimetry. Moreover, dicentrics are known to be lethal in cell proliferation, implying that carcinogenic risk, as discussed by Awa (9), originates mainly from CAs that survive cell division, the so-called stable aberrations.
As such, an increased proliferative/mitotic index does indicate genotoxicity/carcinogenicity of the chemical to which the cells were exposed.
We observed a twofold increase in the frequency of SAs per cell. The difference is significant and may lead to increased probability of chromosomal translocations. The frequency of DG-type associations was highest, whereas the frequency of 3D-type associations was lowest. Similar results were reported earlier for workers exposed to S02 (21) and NH3 (22) and for grape garden workers occupationally exposed to different pesticides (29). Trezepizur et al. (30) found that chromosomes 21 and 22, which are smaller than chromosomes 13, 14, and 15, possess more extended nucleolus organizer regions; consequently, acrocentric chromosomes 21 and 22 enter into associations more frequendy than chromosomes 13, 14, and 15.
A high incidence of SAs has often been considered as a predisposition to an increased tendency of nondisjunction in satellite chromosomes and thus to the induction of D and G trisomies (5). Klossoglu et al. (31) reported cases of increased SAs in families in which such trisomies occur. Among four families in which new cases of translocation trisomics were observed, the trisomics and their parents had an unusually high incidence of SAs (32. Hansson (5) suggested that the tendency of SA is genetically controlled and specific environments may influence the SA. In the present study, NOX in ambient air increased SA in exposed goldsmiths to twice that of controls. The controls were exposed to a background frequency of 0.266 ppb NOX, which is near international standards: World Health Organization, 0.25 ppb (33) and EPA, 0.23 ppb (34).
Nitrous acid (HNO2) deaminates bases in DNA; this is one mechanism for direct mutagenic action. Alkylating agents are formed by nitrosation of primary amines. The formation of N-nitroso compounds from secondary amines and amides is another way for indirect mutagenic activity (35).
Nitrite and nitrate are found in blood after inhalation of NO (36), and nitrosated compounds are formed after inhalation of NO2 (37). Nitrosating agents are formed in the skin when mice are exposed to 50 ppm NO2 for 4 hr; most of these nitrosating agents were derived from cholesterol and some from triglycerides via a peroxidized product (38).
Nitrite derived from nitrate may react in vivo with amines and amides to form Nnitroso compounds, which may have carcinogenic properties. Analysis of urine samples of subjects exposed to nitrates in drinking water revealed the presence of Nnitroso compounds (39). NO2 is also known for its potential to produce free radicals and other potent oxidizing species that may oxidize tissue unsaturated fatty acids. Tabacova (40) suspected that peroxidation of membrane lipid is a primary mechanism for the toxicity of NO2-induced lipid peroxidation in maternal tissues and is related to its embryonic effect.
NO may react intracellularly, or in the medium, with any dissolved oxygen or oxygen-derived radicals that may be generated during exposure to NOX, resulting in the formation of NO2 (41). The combination of NO with NO2 yields a potent nitrosating agent, which has been shown to react with secondary amines to yield carcinogenic N-nitrosamines both in aqueous and lipid phase (42). Two mechanisms for this reaction have been postulated involving either nucleophilic attack by an amine on N203 or a two-step process of radical reaction with the amine (43). While secondary N-nitrosamines generally require metabolic activation for mutagenicity (44), nitrosation of primary amines, as found on DNA bases, results in immediate deamination and DNA codon alteration (45). This latter mechanism may, at least in part, be responsible for the observed mutagenic effects of NO in our system. Deamination of cytosine leads to the formation of uracil, which, if not repaired, causes a base substitution mutation. Likewise, the deamination of methylcytosine results in the formation of a thymine residue in the DNA chain, which is repairable. NO has recently been shown to alter bases in both nucleosides and DNA under aerobic conditions and at physiological pH (46,47).
Accordingly, exposure to NO could result in the expected deamination of cytosine to uracil and account for the observed mutagenicity ofNO.
These results have significant implications for evaluating the genotoxic potential of NOR, especially that of NO, because in most situations involving exposure to high concentrations of NOX, nitric oxide is present in greater concentration relative to NO2. The ability of NO to traverse the cell and sequester in the lipophilic environment within the DNA chain may be crucial to its mutagenic activity (41).

Faculty Position in Environmental Risk Analysis
The University of Californa at Santa Barbara (UCSB) Donald Bren School of Environmental Science and Management i.nvites applications for a tenure-track position at the assistant professor level. We seek an interdisciplinary environmental scientist who conducts research in environmental risks that result from human activities, with a focus on the quantification of probabilities and expected consequences for biological communities, ecosystem processes, or society. Candidates must be able to teach graduate-level courses in all aspects of environmental nrsk ainalysis, including risk identification, estimation, evaluation, and management. Research foci could include pollution, natural resource exploitation, climate change, land use and habitat conversion, or other areas. Postdoctoral or professional experience will be regarded favorably. The Donald Bren School of Environmental Science and Management is a graduate school offering a two-year professional master's degree and an interdisciplinary Ph.D. The curriciulum focuses on climatic, hydrological, ecological, and human systems at local, regional, and global levels, and addresses the consequences of governmental policies, regulations of industrial practice, and corporate strategies. Applicants for faculty positions must have a strong interest in collaborations with faculty and students who conduct interdisciplinary research in environmental science, economics, and policy with implications for environmental problem solving. Our Web page (http',/www.bren.ucsb.edu9) contains more information about the School.
Applications should be sent to Environmental Risk Search Committee, Donald Bren School of Environmental Science and Management, University of California, Santa Barbara, CA 93106-5131 USA, tel: 805-893-7363, fax: 805-893-7612, e-mail: erisk@bren.ucsb.edu. The Ph.D. is normally required at the time of appointment. Applications must include a curriculum vitae, a statement of research interests and teaching philosophy, and copies of three refereed publications. The applicant should arrange for three letters of reference to be sent in support of the application. For fullest consideration, all material should be received by 7 December 1998, although the position will remain open until filled. The start date for the position is 1 July 1999. The University of California is an equal opportunity/affirmative action employer. We encourage all qualified applicants to apply, including minorities, women, and persons with disabilities.