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No increase in chromosome aberrations in lymphocytes from workers exposed to nitrogen fertilisers
Mutation Research, 281 (1992) 133-135 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
133
MUTLET 00621
No increase in chromosome aberrations in lymphocytes from workers exposed to nitrogen fertilisers Armando Rojas Laboratorio de Mutagdnesis, Centro Nacional de lm,estigaciones Cientificas, Cubanacdn, Hacana (Cuba) (Received 7 June 1991) (Revision received 24 September 1991) (Accepted 7 October 1991)
Keywords: Genetic risk; Cytogenetic analysis; Nitrogen fertiliser industry
Summary The putative genetic risk of people occupationally exposed to nitrogen fertilisers was studied using the structural chromosome aberration assay in peripheral blood lymphocytes. The exposed group included 23 subjects working at complex and mixed fertiliser plants. The percent of aberrant cells (Ab.C %) and break to cell ratio ( B / C ) were 0.95% and 0.01 respectively. The matched control group (20 subjects) was found to have 0.80% Ab.C and a B / C ratio of 0.0085. The results show a lack of detectable genetic damage in exposed people using this cytogenetic approach.
There is a considerable amount of experimental evidence indicating that N-nitroso compounds are potent carcinogens (IARC, 1978). Such compounds can form by reaction of nitrosable amines or amides with nitrite. Endogenous formation of N-nitroso compounds can occur following ingestion of large amounts of nitrate, and contact with nitrated sources. Another important, but not well understood, mechanism is via inhalation of nitrogen oxides which has been correlated with excretion of N-nitrosodimethylamine in the urine (Tannenbaum, 1987). Several authors have postulated a relationship between endogenous formation of N-nitroso
Correspondence: Dr. A. Rojas, Laboratorio de Mutag6nesis, Centro Nacional de Investigaciones Cientificas, Apartado 6880, Cubanac~n, Havana (Cuba).
compounds and increased risk for gastric cancer (Hill, 1979; Young, 1980). Specifically, different epidemiological studies have shown a positive correlation between gastric cancer and nitrogen fertiliser usage (Armijo and Coulson, 1975). However, in some cases, no clear correlations have been shown concerning the aetiological role of these compounds in any human cancer (Fraser et al., 1980). A large amount of evidence on endogenous synthesis of N-nitroso compounds demonstrated the excretion of nitroalkylamines in urine and even alkylated nucleic acid bases (Lu et al., 1987; Spiegelhalder et al., 1987; Shuker et al., 1987). However, genetic monitoring studies in humans are still rather scanty. Recently, Miller (1984) failed to show any effect of ingested nitrite on the level of unscheduled DNA synthesis in leukocytes from volunteers after consumption of amine and nitrite sources.
134
Taking into account that nitrogen fertiliser workers are mainly exposed to nitrate, nitrogen oxides a n d / o r nitrated sources and the very scarce data concerning biological monitoring of such populations, a cytogenetic analysis was performed in order to evaluate the consequences of such exposure by studying structural chromosome aberrations.
and the break to cell ratio (B/C). The intergroup differences were statistically evaluated using Student's t-test. Analysis of air samples was also done to measure SO 2, NH 3 and NO 2 levels in the work-place air. Moreover, the level of total dust in air, mainly formed by fertiliser particulate aerosols, was also measured.
Materials and methods
Results and discussion
Twenty=three workers at mixed and complex nitrogen fertiliser plants were selected, who had not been on drug treatment or had recent viral infections or X-rays during 3 months before examination. The mean age was 33.2 + 5.8 years. The control group comprised 20 subjects, with no known chemical exposure at work. The mean age was 36.5 _+ 6.1 years. The numbers of smokers in exposed and control groups represented 52% (12 out of 23) and 55% (11 out of 20), respectively. The cytogenetic analysis was performed as follows: blood samples were taken using heparinised syringes. Whole blood microcultures were done. Lymphocytes were grown in MEM (Gibco) supplemented with foetal calf serum 20% (Gibco) and stimulated for 50 h by phytohaemagglutinin (PHA 15, Wellcome). All slides were coded and scored blind. A hundred metaphases were analysed for each subject. A cell bearing at least a break or exchange was considered aberrant. Gaps, defined as unstained regions with a length less than the width of one chromatid in the same chromosome, were also registered but not included for calculating the percentage of ceils with aberrations (Ab.C %)
Nitrogen fertiliser production involves exposure to various nitrate sources, so it may potentially lead to endogenous formation of N-nitroso compounds. The results of the cytogenetic analysis, given in Table 1, failed to show any differences between exposed workers and control subjects. All values obtained for SO 2, NH 3 and NO 2 were below the corresponding Cuban maximal allowable concentrations (MAC) of 20 m g / m 3, 20 m g / m 3, and 10 m g / m 3, respectively. On the other hand, the level of total dust (mainly formed by fertiliser particulate aerosols) reached values around MAC level (4 m g / m 3) and occasionally slightly higher values. Several factors should be taken into consideration to explain our negative findings. Endogenous formation is limited by the pharmacokinetic parameters of nitrate, its low conversion level into nitrite by oral microflora (Eisenbrand et al., 1980; Ellen et al., 1982) as well as the low pH values required or enzymatic catalysis by intestinal bacteria at neutral pH (Archer et al., 1982). Also the nitrite level in the blood is maintained very low because nitrite is rapidly oxidised to nitrate by the action of oxyhaemoglobin (Rodney, 1976).
TABLE 1 RESULT OF THE C Y T O G E N E T I C ANALYSIS Group
N
Gaps
B'
B"
E
% AC ~'
B / C ~'
Exposed
23
19 (0-3)
15 (0-2)
6 (0-1)
1 -
0.95 (0-2.0)
0.01 (0-0.02)
Control
20
12 (0-2)
10 (0-2)
5 (0-1)
1 -
0.80 (0-2.0)
0.0085 (0-0.02)
B', chromatid breaks; B", chromosome breaks; E, exchanges; % AC, percent of aberrant cells; B / C , break to cell ratio. " Calculated from individual means. Ranges are shown in parentheses.
135
Finally, 2 other aspects could have special relevance: the rapid metabolic decay of some Nnitroso compounds formed in vivo and the metabolic activation needed to transform them into active metabolites (Lijinsky, 1976). Though some nitroalkylamines reach to bloodstream, it seems that lymphocytes have not enough metabolic activation capacity to reduce them to active metabolites, as shown in studies in vitro with p e r i p h e r a l blood lymphocytes (Sankaranarayanan, 1981). Taking into account that cytogenetic analysis failed to show any differences between exposed and control groups, and considering the very special features of the in vivo nitrosation process, further studies should be done, including analytical procedures for determining N-nitroso compounds in body fluids and putative precursors in the work-place air. Besides these analytical approaches, analysis of the mutagenicity of urine and work-place air using Ames test Salmonella strains will also be very convenient. References Archer, M.C., L. Lee and W.R. Bruce (1982) Analysis and formation of nitrosamines in the human intestine, in: H. Bartsch, 1.K. O'Neill, M. Castegnaro and M. Okada (Eds.), N-Nitroso Compounds: Occurrence and Biological Effects, IARC Sci. Publ. No. 14, pp. 357-363. Armijo, R., and A.H. Coulson (1975) Epidemiology of stomach cancer in Chile. The role of nitrogen fertilisers, Int. J. Epidemiol., 4, 301-309. Eisenbrand, G., B. Spiegelhalder and R. Preussmann (1980) Nitrate and nitrate in saliva, Oncology, 37, 227-232. Ellen, G., P.L. Schuller, E. Bujns, P.G.A.M. Froeling and H. Baadenhuijsen (1982) Volatile N-nitrosamines, nitrate and nitrite in urine and saliva of healthy volunteers after administration of large amounts of nitrate, in: H. Bartsch, I.K. O'Neill, M. Castegnaro and M. Okada (Eds.), NNitroso Compounds: Occurrence and Biological Effects, IARC Sci. Publ. No. 14, pp. 365-378. Fraser, P., C. Chilvers, V. Beral and M.J. Hill (1980) Nitrate and human cancer. A review of the evidence, Int. J. Epidemiol., 9, 3-11. Hill, M.J. (1979) In vivo bacterial N-nitrosation and its possible role in human cancer, in: E.C. Miller, J.A. Miller, I. Hirono, T. Sugimura and S. Takayama (Eds.), Naturally Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis, Proc. 9th Int. Symp. Princess Takamatsu Res.
Fund., Baltimore University Park Press, Tokyo, pp. 229240. IARC (1978) IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vol. 17, Some Nnitroso compounds, International Agency for Research on Cancer, Lyon. Lijinsky, W. (1976) Carcinogenic and mutagenic N-nitroso compounds, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Plenum, New York, pp, 193-217. Lu, S.H., W.X. Yang, L.P. Guo, F.M. Li, G.J. Wang, J.S. Zhang and P.Z. Li (1987) Determination of N-nitrosamines in gastric juice and urine and a comparison of endogenous formation of N-nitrosoproline and its inhibition in subjects from high- and low-risk areas for oesophageal cancer, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds.), The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mechanisms, IARC Sci. Publ. No. 84, pp. 538-543. Miller, C.T. (1984) Unscheduled DNA synthesis in human leukocytes after a fish (amine source) meal with or without salad (nitrite source), in: I.K. O'Neill, R.C. von Borstel, C.T. Miller, J. Long and H. Bartsch (Eds.), N-Nitroso Compounds: Occurrence, Biological Effects and Relevance to Human Cancer, IARC Sci. Publ. No. 57, pp. 609-613. Rodney, F.L. (1976) A mechanism for the conversion of oxyhemoglobin to methemoglobin by nitrite, Clin. Chem., 22, 1986-1990. Sankaranarayanan, K. (1981) Comparative mutagenicity of dimethylnitrosamine and diethylnitrosamine, in: F.J. de Serres and M.D. Shelby (Eds.), Comparative Chemical Mutagenesis, Plenum, New York, pp. 787-856. Shuker, D.E.G., E. Bailey and P.B. Farmer (1987) Excretion of methylated nucleic acid bases as an indicator of exposure to nitrosable drugs, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds3, The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mechanisms, IARC Sci. Publ. No. 84, pp. 407-410. Spiegelhalder, B., J. Muller, H. Drasche and R. Preussmann (1987) N-Nitrosodiethanolamide excretion in metal grinder, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds.), The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mechanisms, IARC Sci. Publ. No. 84, pp. 550-552. Tannenbaum, S.R. (1987) Endogenous formation of N-nitroso compounds. A current perspective, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds.), The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mechanisms, IARC Sci. Publ. No. 84, pp. 292-296. Young, C.S. (1980) Research on esophageal cancer in China: a review, Cancer Res., 40, 2633-2644.
Communicated by S.M. Galloway
133
MUTLET 00621
No increase in chromosome aberrations in lymphocytes from workers exposed to nitrogen fertilisers Armando Rojas Laboratorio de Mutagdnesis, Centro Nacional de lm,estigaciones Cientificas, Cubanacdn, Hacana (Cuba) (Received 7 June 1991) (Revision received 24 September 1991) (Accepted 7 October 1991)
Keywords: Genetic risk; Cytogenetic analysis; Nitrogen fertiliser industry
Summary The putative genetic risk of people occupationally exposed to nitrogen fertilisers was studied using the structural chromosome aberration assay in peripheral blood lymphocytes. The exposed group included 23 subjects working at complex and mixed fertiliser plants. The percent of aberrant cells (Ab.C %) and break to cell ratio ( B / C ) were 0.95% and 0.01 respectively. The matched control group (20 subjects) was found to have 0.80% Ab.C and a B / C ratio of 0.0085. The results show a lack of detectable genetic damage in exposed people using this cytogenetic approach.
There is a considerable amount of experimental evidence indicating that N-nitroso compounds are potent carcinogens (IARC, 1978). Such compounds can form by reaction of nitrosable amines or amides with nitrite. Endogenous formation of N-nitroso compounds can occur following ingestion of large amounts of nitrate, and contact with nitrated sources. Another important, but not well understood, mechanism is via inhalation of nitrogen oxides which has been correlated with excretion of N-nitrosodimethylamine in the urine (Tannenbaum, 1987). Several authors have postulated a relationship between endogenous formation of N-nitroso
Correspondence: Dr. A. Rojas, Laboratorio de Mutag6nesis, Centro Nacional de Investigaciones Cientificas, Apartado 6880, Cubanac~n, Havana (Cuba).
compounds and increased risk for gastric cancer (Hill, 1979; Young, 1980). Specifically, different epidemiological studies have shown a positive correlation between gastric cancer and nitrogen fertiliser usage (Armijo and Coulson, 1975). However, in some cases, no clear correlations have been shown concerning the aetiological role of these compounds in any human cancer (Fraser et al., 1980). A large amount of evidence on endogenous synthesis of N-nitroso compounds demonstrated the excretion of nitroalkylamines in urine and even alkylated nucleic acid bases (Lu et al., 1987; Spiegelhalder et al., 1987; Shuker et al., 1987). However, genetic monitoring studies in humans are still rather scanty. Recently, Miller (1984) failed to show any effect of ingested nitrite on the level of unscheduled DNA synthesis in leukocytes from volunteers after consumption of amine and nitrite sources.
134
Taking into account that nitrogen fertiliser workers are mainly exposed to nitrate, nitrogen oxides a n d / o r nitrated sources and the very scarce data concerning biological monitoring of such populations, a cytogenetic analysis was performed in order to evaluate the consequences of such exposure by studying structural chromosome aberrations.
and the break to cell ratio (B/C). The intergroup differences were statistically evaluated using Student's t-test. Analysis of air samples was also done to measure SO 2, NH 3 and NO 2 levels in the work-place air. Moreover, the level of total dust in air, mainly formed by fertiliser particulate aerosols, was also measured.
Materials and methods
Results and discussion
Twenty=three workers at mixed and complex nitrogen fertiliser plants were selected, who had not been on drug treatment or had recent viral infections or X-rays during 3 months before examination. The mean age was 33.2 + 5.8 years. The control group comprised 20 subjects, with no known chemical exposure at work. The mean age was 36.5 _+ 6.1 years. The numbers of smokers in exposed and control groups represented 52% (12 out of 23) and 55% (11 out of 20), respectively. The cytogenetic analysis was performed as follows: blood samples were taken using heparinised syringes. Whole blood microcultures were done. Lymphocytes were grown in MEM (Gibco) supplemented with foetal calf serum 20% (Gibco) and stimulated for 50 h by phytohaemagglutinin (PHA 15, Wellcome). All slides were coded and scored blind. A hundred metaphases were analysed for each subject. A cell bearing at least a break or exchange was considered aberrant. Gaps, defined as unstained regions with a length less than the width of one chromatid in the same chromosome, were also registered but not included for calculating the percentage of ceils with aberrations (Ab.C %)
Nitrogen fertiliser production involves exposure to various nitrate sources, so it may potentially lead to endogenous formation of N-nitroso compounds. The results of the cytogenetic analysis, given in Table 1, failed to show any differences between exposed workers and control subjects. All values obtained for SO 2, NH 3 and NO 2 were below the corresponding Cuban maximal allowable concentrations (MAC) of 20 m g / m 3, 20 m g / m 3, and 10 m g / m 3, respectively. On the other hand, the level of total dust (mainly formed by fertiliser particulate aerosols) reached values around MAC level (4 m g / m 3) and occasionally slightly higher values. Several factors should be taken into consideration to explain our negative findings. Endogenous formation is limited by the pharmacokinetic parameters of nitrate, its low conversion level into nitrite by oral microflora (Eisenbrand et al., 1980; Ellen et al., 1982) as well as the low pH values required or enzymatic catalysis by intestinal bacteria at neutral pH (Archer et al., 1982). Also the nitrite level in the blood is maintained very low because nitrite is rapidly oxidised to nitrate by the action of oxyhaemoglobin (Rodney, 1976).
TABLE 1 RESULT OF THE C Y T O G E N E T I C ANALYSIS Group
N
Gaps
B'
B"
E
% AC ~'
B / C ~'
Exposed
23
19 (0-3)
15 (0-2)
6 (0-1)
1 -
0.95 (0-2.0)
0.01 (0-0.02)
Control
20
12 (0-2)
10 (0-2)
5 (0-1)
1 -
0.80 (0-2.0)
0.0085 (0-0.02)
B', chromatid breaks; B", chromosome breaks; E, exchanges; % AC, percent of aberrant cells; B / C , break to cell ratio. " Calculated from individual means. Ranges are shown in parentheses.
135
Finally, 2 other aspects could have special relevance: the rapid metabolic decay of some Nnitroso compounds formed in vivo and the metabolic activation needed to transform them into active metabolites (Lijinsky, 1976). Though some nitroalkylamines reach to bloodstream, it seems that lymphocytes have not enough metabolic activation capacity to reduce them to active metabolites, as shown in studies in vitro with p e r i p h e r a l blood lymphocytes (Sankaranarayanan, 1981). Taking into account that cytogenetic analysis failed to show any differences between exposed and control groups, and considering the very special features of the in vivo nitrosation process, further studies should be done, including analytical procedures for determining N-nitroso compounds in body fluids and putative precursors in the work-place air. Besides these analytical approaches, analysis of the mutagenicity of urine and work-place air using Ames test Salmonella strains will also be very convenient. References Archer, M.C., L. Lee and W.R. Bruce (1982) Analysis and formation of nitrosamines in the human intestine, in: H. Bartsch, 1.K. O'Neill, M. Castegnaro and M. Okada (Eds.), N-Nitroso Compounds: Occurrence and Biological Effects, IARC Sci. Publ. No. 14, pp. 357-363. Armijo, R., and A.H. Coulson (1975) Epidemiology of stomach cancer in Chile. The role of nitrogen fertilisers, Int. J. Epidemiol., 4, 301-309. Eisenbrand, G., B. Spiegelhalder and R. Preussmann (1980) Nitrate and nitrate in saliva, Oncology, 37, 227-232. Ellen, G., P.L. Schuller, E. Bujns, P.G.A.M. Froeling and H. Baadenhuijsen (1982) Volatile N-nitrosamines, nitrate and nitrite in urine and saliva of healthy volunteers after administration of large amounts of nitrate, in: H. Bartsch, I.K. O'Neill, M. Castegnaro and M. Okada (Eds.), NNitroso Compounds: Occurrence and Biological Effects, IARC Sci. Publ. No. 14, pp. 365-378. Fraser, P., C. Chilvers, V. Beral and M.J. Hill (1980) Nitrate and human cancer. A review of the evidence, Int. J. Epidemiol., 9, 3-11. Hill, M.J. (1979) In vivo bacterial N-nitrosation and its possible role in human cancer, in: E.C. Miller, J.A. Miller, I. Hirono, T. Sugimura and S. Takayama (Eds.), Naturally Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis, Proc. 9th Int. Symp. Princess Takamatsu Res.
Fund., Baltimore University Park Press, Tokyo, pp. 229240. IARC (1978) IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vol. 17, Some Nnitroso compounds, International Agency for Research on Cancer, Lyon. Lijinsky, W. (1976) Carcinogenic and mutagenic N-nitroso compounds, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Plenum, New York, pp, 193-217. Lu, S.H., W.X. Yang, L.P. Guo, F.M. Li, G.J. Wang, J.S. Zhang and P.Z. Li (1987) Determination of N-nitrosamines in gastric juice and urine and a comparison of endogenous formation of N-nitrosoproline and its inhibition in subjects from high- and low-risk areas for oesophageal cancer, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds.), The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mechanisms, IARC Sci. Publ. No. 84, pp. 538-543. Miller, C.T. (1984) Unscheduled DNA synthesis in human leukocytes after a fish (amine source) meal with or without salad (nitrite source), in: I.K. O'Neill, R.C. von Borstel, C.T. Miller, J. Long and H. Bartsch (Eds.), N-Nitroso Compounds: Occurrence, Biological Effects and Relevance to Human Cancer, IARC Sci. Publ. No. 57, pp. 609-613. Rodney, F.L. (1976) A mechanism for the conversion of oxyhemoglobin to methemoglobin by nitrite, Clin. Chem., 22, 1986-1990. Sankaranarayanan, K. (1981) Comparative mutagenicity of dimethylnitrosamine and diethylnitrosamine, in: F.J. de Serres and M.D. Shelby (Eds.), Comparative Chemical Mutagenesis, Plenum, New York, pp. 787-856. Shuker, D.E.G., E. Bailey and P.B. Farmer (1987) Excretion of methylated nucleic acid bases as an indicator of exposure to nitrosable drugs, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds3, The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mechanisms, IARC Sci. Publ. No. 84, pp. 407-410. Spiegelhalder, B., J. Muller, H. Drasche and R. Preussmann (1987) N-Nitrosodiethanolamide excretion in metal grinder, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds.), The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mechanisms, IARC Sci. Publ. No. 84, pp. 550-552. Tannenbaum, S.R. (1987) Endogenous formation of N-nitroso compounds. A current perspective, in: H. Bartsch, I. O'Neill and R. Schulte-Herman (Eds.), The Relevance of N-Nitroso Compounds to Human Cancer: Exposure and Mechanisms, IARC Sci. Publ. No. 84, pp. 292-296. Young, C.S. (1980) Research on esophageal cancer in China: a review, Cancer Res., 40, 2633-2644.
Communicated by S.M. Galloway