Introduction

Metals are the ubiquitous chemical entities, known to cause mutations in a variety of test systems. Based on the epidemiological studies, some of them are classified as human carcinogen (Sunderman., [1] 1984; IARC, [2] 1990) as well as mutagens and the human exposure to these metallic compounds was found among the workers in a large number of professional groups. The occupational exposure to chromium compound was typically found in stainless steel welders, (IARC, 1993) chrome platers (Gambelunghe [3] et al., 2003), leather tanning and chromate production workers (Langard., [4] 1990).

Leather tanning is an age-old practice in India, which is recently recognized as one of the potential polluting industry of considerable importance. The wastes from tannery contain an excess amount of chromium ranging from 100-200 mg/l and the spent chrome liquor contains 2900 – 4500 mg/l (Nandakumar [5] and Baekavathy, 1986). Chronic exposure, poor working conditions, lack of civic sense and awareness of the potential hazard leads to an occupational health hazardous situations at an industrial set up.

Chromium compounds were known to be potent carcinogens (IARC, [6] 1987) and mutagens (Alcedo and Wetterhahn [7] , 1990), which can induce a spectrum of DNA damage (Xu [8] et al., 1992), gene mutation (Deflora et al., 1990), sister chromatid exchanges (Montaldi [9] et al., 1987) and chromosomal aberrations (Wise [10] et al., 1992). Several workers already reported the mutagenic and carcinogenic potentialities of chromium compound in bacterial and mammalian cell-based mutagenicity assays (Deflora, 1990; Snow, [11] 1992; Stearns [12] et al., 2002; Quievryn [13] et al., 2003). The investigations carried out to examine the genotoxicity in workers occupationally exposed to chromium are meager. (Deng et al., 1988; Gennart [14] et al., 1993, Jelmert [15] et al., 1994; Werfel [16] et al., 1998 and Burgaz [17] et al., 2002). However, there are reports on positive genotoxic effects in populations exposed to chromium (Sarto et al., 1982; Vaglenov [18] et al., 1999; Wu et al., 2000 and Benova [19] et al., 2002) also a negative findings (Zhitkovich [20] et al., 1996 and Huvinen [21] et al., 2002). The studies on the evaluation of genetic damage in chrome tanning workers exposed to chromium are scanty and rather conflicting. Hence an attempt was made during this study to evaluate the mutagenic potential of chromium compound in occupationally exposed leather tanning industrial workers in a tanning industry situated at Bakaram industrial area, Hyderabad, Andhra Pradesh, India by using the standard cytogenetic protocol of chromosome aberrations.

Materials and Methods

Air and Blood Chromium analysis: Ambient air samples at different locations of the industry were collected to evaluate chromium levels. The samples were collected on membrane filter (37mm, 0.8μm pore size) and in 1M HNO3 using KIMOTO air samples at a flow rate of 1-2 LPM for 8hrs in day shift. The collected samples were wet digested with concentrated HNO3 and analyzed for chromium by using atomic absorption spectrophotometer (Double Beam 3100 model, Perkin Elmer, USA)(Petering [22] et al., 1993). Blood samples were collected in heparinized vials and were wet digested in microwave digestive system using conc. HNO3 and were analyzed for the metal by atomic absorption spectrophotometer (Double Beam 3100 model, Perkin Elmer, USA)(Petering et al., 1993).

Study population: 160 tannery industrial workers (80 each of smoker and non-smoker group) were selected from a leather tanning industry situated at Bakaram industrial area, Hyderabad, Andhra Pradesh , India, to evaluate the genotoxic potential of Chromium compound by using the standard cytogenetic protocol of chromosomal aberrations in peripheral blood lymphocytes . The selected workers belong to the age group of 25-50 years and belong to the same socio-economic status. Simultaneously 120 individuals. (62 smoker and 58 non-smoker group) who didn’t have any history of exposure, were also selected for comparison (control). The data was further analyzed on the basis of their personnel habitat into smokers and non-smokers and also on their duration of service into 1-5. 6-10 and 11-15 years, respectively. All participants were informed about the objectives of the investigation and written consent was obtained from each person. Personnel data and family histories were collected by the interviews and questionnaires. This study was approved by institutional ethics committee.

Lymphocyte isolation and Cell culture: Blood samples were collected by vein puncture in Heparinized centrifuge test tubes and transported to the laboratory within 2-4 hrs. Lymphocytes were isolated by gradient centrifugation and washed three times in phosphate buffer saline. Lymphocytes from each individual sample were divided into several aliquots. One part was used to prepare 2 ml lymphocyte cultures, with a cell density of 1x 106/ ml in RPMI – 1640 culture media , supplemented with 15% fetal calf serum , antibiotic and 1% phytohaemagglutinin. The lymphocyte cultures were incubated at 37°C in a humid atmosphere with 5 % CO2 for 48 hrs.

Cell harvesting and slide preparation: Cell harvesting was done followed by colchicine treatment (0.1 μg/ml culture medium) for the last 4 hours of the 48 hours incubation, hypotonic treatment in prewarmed 0.075M KCl solution for 15 minutes at 37°C and three fixations in chilled methanol – acetic acid mixture (v/v, 3:1). The cells were dropped on to cool wet slides. The slides were air dried and stained in 2% Giemsa stain (pH 6.8) mounted with DPX and scored for chromosomal aberrations by adopting the method of (Moorhead [23] et al., 1960).

Aberration scoring and statistical analysis: Chromosomal aberrations were scored on coded slides by two independent observers using research microscope under 10x and 100x oil immersion. One hundred metaphases per subject were scored in the heavy metal exposure study. Only well spread metaphases with 46 centromeres were selected. Gaps (A chromatic lesions) were counted separately but not included in the frequency of the cells with aberrations. The frequencies of the cells with aberrations were tested statistically by using a Chi-square (2x2 contingency) test to find out the significant levels between the groups tested.

Results

The result on chromium concentrations in the ambient air and in the blood was depicted in (Table-I). The levels of chromium were found in higher concentrations at tanning unit (43.26 ± 20.20μg/m3) Spray dyer (22.14 ± 10.60 μg/m3) and in administrative office (3.86 ± 0.61 μg/m3).The chromium levels observed are much lower than threshold limit value of 500 μg/ m3.The mean blood chromium levels in the exposed workers was 6.83 ± 1.32 μg/100ml which was considered higher when compared to the control value of 2.67 ± 0.34 μg/100ml.

The overall result on the incidence of chromosomal aberrations in leather tanning industrial workers are presented in (Table-II and III). An increased pattern in the frequency of chromosomal aberration was observed in the chromium exposed group when compared to the control. As a result of this the percentage of total chromosomal aberrations got increased from 6.20 ± 0.12 in the exposed workers as against 1.61 ± 0.09 in the control subjects.

In order to monitor the longitudinal variations of chromosomal aberrations, further analysis was carried out on the basis of duration of employment and on the basis of their smoking habit. A gradual increase in the frequency of total chromosomal aberrations of 4.82 ± 0.33, 6.84 ± 0.73 and 8.22 ± 1.38 were observed with the increase in the duration of exposure of 1-5, 6-10, 11-15 years respectively(Table-II) Similar trend was observed among the smoker and non-smoker subjects. A 5.35 ± 0.36, 7.76 ± 0.55 and 10.80 ± 0.84 increased percentage of chromosomal aberrations were observed among the smoker exposed group as the increase in the duration of exposure at the industry as against the control smoker group of 3.70 ± 0.30, 5.92 ± 0.48 and 8,40 ± 0.74 respectively (Table-III).

Discussion

Large number of industries releases the chromium compound into the air, water and soil. In the air this compound is mainly present in the form of fine dust (Rowbotham [24] et al., 2000). The permissible exposure limits for chromium in work place during an 8hr, 40hrs work weak is 100μg/m3 and the recommended exposure limit is 1μg/m3. The investigated result is supported by the observations made by (Sathwara [25] et al., 2007) in a chemical based industry.

There is a clear evidence that some metals represent a carcinogenic hazard to man and several metallic compounds has been identified as human carcinogens (Friberg [26] et al.,1986; IARC,[27] 1993). Evaluation of mutagenic hazards has become an integral part in the toxicological assessment with a number of environmental chemical (Dearfield [28] et al., 1991) The cytogenetic methods was routinely employed in monitoring the populations, exposed to industrial chemicals (De Jong [29]et al., 1988) . A significant increase in the frequency of chromosomal aberrations among the workers of leather tanning industry is once again establishing the mutagenic nature of the chromium compound, as the tannery effluents have the potential to damage the DNA of test organisms (O’Brien et al., 2003).

Induction of chromosomal aberrations in human peripheral blood lymphocytes by chromate compounds were reported earlier (Newton [30] and Lilly, 1986) .However the present results were attributed to the observations made in peripheral blood lymphocytes of chromate workers, exposed to chromium containing fumes (Stella [31] et al., 1982 ; Sarto [32] et al., 1982; Deng [33] et al., 1983; Deng [34] et al., 1988; Koshi [35] et al., 1984). Further the result were comparable with that of the observations among the workers exposed to benzene pyrene epoxides (Jelmert et al., 1994; Wei [36] et al., 1996) ; pesticides (Major [37] et al., 1998).

The chromosomal aberrations observed in the study was mainly of chromatid type, which can be capable to induce more number of aberrations in late S1 phase or early G2 phase of the cell cycle (Rita [38] et al., 1987). The presence of less iso-chromatid aberrations may reflect a direct effect of the compound on G1 phase. Further, the aberrations recorded even after 11-15 yrs of exposure may be due to the phenomenon of aging of the cells in circulating blood lymphocytes.

A significant increase in chromosomal aberrations in smoker and non-smoker exposed group to chromium compound than their respective controls was due to the effect of cigarette smoke on the genetic material. Even the synergistic interaction would also be possible, but the actual mechanism is not yet to be documented. Similar findings were reported by several workers among the smoker groups, occupationally exposed to rubber, (Sorsa [39] et al., 1983; Prasad [40] and Reddy, 1993); pesticides (Linnainmaa, [41] 1983) ; heavy metals (Liu [42] and Dixion, 1996; Wu [43] et al., 2000) and plastic workers (Van-hummelen [44] et al., 1994), which will support the present investigation.

The chromate compounds are well known as human and animal carcinogens (Deflora [45, 46], 2000) and the workers occupationally exposed to chromium compound are prone towards the increased risk of cancer (Gibb [47] et al., 2000). The exact mechanism for chromium mutagenecity has not been so far reported, but it has been suggested that the chromium compound give off hydroxyl, cysteinyl and thionyl radicals (Stearns [48] and Wetterhahn, 1994; Izzotti [49] et al., 1998) during cellular reductions. These radicals can interact directly with DNA – chromatin to form DNA single strand breaks, DNAprotein cross links, chromium-DNA adducts and DNA-DNA cross links(Sugiyama [50] et al., 1991 and Manning [51] et al., 1992), ultimately leads to chromosomal breakage and mutations (Hodges [52] et al., 2001 and O-Brien [53] et al., 2003).

Conclusion

In conclusion, the study reveals that there is a significant increase in the frequency of chromosomal aberrations in the peripheral blood lymphocytes of leather tanning industrial workers. This increase may be due to chromosomal instability and also the fact that chromium being used in tannery industry is a potent mutagen. Further this study needs an elaborate and exhaust study in order to interpret the data in a conceptual frame work. The present study facilitates to make a decision on issue of pollution control, mutagenesis and risk analysis. It is also helpful to have a solution for the damage caused by the polluting industries.

Acknowledgments

We are thankful to the participants and the industrial authorities for their support and cooperation. The authors express their sincere thanks to the University authorities for providing the necessary facilities.

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  Table 1: *Values in the ambient air at different work stations was less than the threshold values of 500 μg/m3.

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  Table 2: 100 metaphases were scored for each sample
  Gaps and polyploids are not included in aberrant cells.
  Values in parenthesis are percentages ± S.E

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  Table 3: 100 metaphases were scored for each sample.
  Values in parenthesis are percentages ± S.E.
  Gaps and polyploidy are not included in aberrant cells.
  * P< 0.05 and **P< 0.01.