Mutation Research, 277 (1992) 1-9 © 1992 El'sevier Science Publishers B.V. All rights reserved 0165-1110/92/$15.00
1
MUTREV 07314
Genetic toxicology of propylene oxide and trichloropropylene oxide a review A.K. Giri Division of Toxicology, Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226001 (India) (Received 25 July 1991) (Revision received 17 October 1991) (Accepted 17 October 1991)
Keywords: Propylene oxide; Trichloropropylene oxide; Genotoxicity
Summary Aliphatic epoxides are a group of compounds extensively used in industry and as laboratory reagents, and can be produced as metabolic intermediates. An important aliphatic epoxide is propylene oxide, extensively used in the production of propylene glycol and polyols used in the manufacture of polyurethane polymers. The widespread human exposure to propylene oxide is of great health concern. In this review an attempt has been made to evaluate and update the genotoxic effects of propylene oxide and trichloropropylene oxide based on the available literature.
Aliphatic epoxides are widely used in industry and as laboratory reagents. Some of the aliphatic epoxides are highly toxic and some are also animal carcinogens. Because of the extensive use of epoxides, questions have been raised concerning their genotoxic potential (Manson, 1980; Ehrenberg and Hussain, 1981). An important aliphatic epoxide is propylene oxide (PO). PO is extensively used in the production of propylene glycol and polyols. Human populations are exposed mainly in industrial operations using PO. Polyols are used in the manufacture of polyurethane polymers. Propylene glycol, made
Correspondence: Dr. A.K. Giri, Scientist, Division of Toxicology, Central Drug Research Institute, Chattar Manzil Palace, Post Box 173, Lucknow 226001 (India). The C.D.R.I. communication number for this paper is 4877.
by the reaction of PO with water, is mainly used in the production of unsaturated polyester resins (IARC, 1985). PO is also used for sterilizing packaged food products in fumigation chambers (Berg, 1984). The U.S. Food and Drug Administration (1980) has approved the use of PO as a direct or indirect food additive, provided it does not exceed specified limits. There is sufficient evidence that PO is carinogenic in rodents. In an oral gavage study in rats, a dose-dependent increase was found in the incidence of local tumours, mainly squamous-cell carcinomas of the forestomach (Dunkelberg, 1982). In an inhalation study in mice and rats, PO produced haemangiomas and haemangiosarcomas of the nasal turbinates in mice and papillary adenomas of the nasal turbinates in rats (National Toxicology Program, 1984; Lynch et al., 1984a). PO also induced local fibrosarcomas after
subcutaneous administration to mice (Dunkelberg, 1981). During the past 25 years there has been an increasing number of reports on the genotoxicity and interaction with DNA of PO. Because of extensive occupational exposure to humans, much attention has been given to this compound. We have been testing the short-term genotoxic effects of different industrial chemicals including aliphatic epoxides and food additives (Giri, 1986; Giri et al., 1986a,b; Mukherjee et al., 1988; Giri and Que Hee, 1988; Roychaudhury and Giri, 1989; Giri et al., 1989, 1990). The mutagenic and genotoxic effects of PO and trichloropropylene oxide (TCPO) are reviewed.
Short-term mutagenicity assay The mutagenic effects of PO on microorganisms indicate that PO is a direct-acting mutagen which induces base-pair substitutions. It did not induce mutation in T2 bacteriophage (Cookson et al., 1971). Several authors have reported that PO is mutagenic in Salmonella typhimurium strains TA1535 and TA100 both with and without metabolic activation. It was also mutagenic in Bacillus subtilis and Escherichia coli (Table 1). Pfeiffer and Dunkelberg (1980) observed a dose-dependent increase in the number of revertants in Salmonella strains TA100 and TA1535 (PO 10-200/.~mole/plate) without metabolic activation. They found that TA100 was less sensitive than TA1535. No mutagenicity was observed in strains TA98 and TA1537. Similar results were reported by McMahon et al. (1979) and DeFlora (1981) with positive effects in TA1535 and TA100 but negative results in TA1537, TA1538 and TA98. Garro and Phillips (1980) described a mutagenesis assay which detects the induction of forward mutations in isolated DNA. The assay utilizes replicative form DNA of the temperate Bacillus subtilis phage q~105 and tests the ability of chemicals to induce lesions which inactivate phage genes involved in lysogen formation. PO (25-100 mM) was reacted with single-stranded qq05 DNA of B. subtilis in dimethylformamide buffer at 42°C for 30 min. The presence of mutagenic lesions in the treated DNA sample was detected by marker rescue with both the Hcr +
and H c r - recipient strains. The induction of clear plaque-forming units was significant with both strains. No consistent decrease in marker rescue activity was observed although there was a decrease in the frequency of clear plaque-forming units generated with the Hcr + host at the highest PO concentration (Garro and Phillips, 1980). Hemminki and Falck (1979) studied the mutagenicity and alkylation rate of reaction with 4(p-nitrobenzyl) pyridine for both PO and TCPO. They observed mutagenicity in Salmonella strain TA100 and E. coli WP2 uvrA for both compounds. A good correlation was observed in the mutagenicity and alkylation rate in the case of TCPO, but PO showed relatively less mutagenicity than its reactivity with 4-(p-nitrobenzyl) pyridine. These structurally related base-pair substitution mutagens do not require metabolic activation and Callen and Ong (1978) reported that the genetic activity of TCPO could be eliminated or reduced in the presence of the $9 fraction of liver homogenate. This can be explained by the rapid detoxication by glutathione and the rapid binding of TCPO to epoxide hydrolase and other proteins within the $9 fraction (Sinsheimer et al., 1987). This would result in a partial or complete absence of unbound TCPO able to penetrate the target cell and bind to DNA. The induction of mutation by PO in isolated transforming DNA of Bacillus subtilis was also reported by Phillips et al. (1980). They also studied the capacity of PO to inactivate transforming activity and disrupt gene linkage in treated DNAs. Although PO was a weak mutagen in this assay, it produced more extensive disruption of gene linkage at the concentration which produced comparable levels of inactivation of transforming activity. Rosman et al. (1987) also reported the mutagenic effects of several propylene oxide derivatives including 1-naphthyl propylene oxide (NPO) in Salmonella strains TA100 and TA1535 using both the preincubation and plate incorporation tests. We have also studied the mutagenicity of NPO and TCPO in preincubation and plate tests in Salmonella strain TA100 without $9. Chemical reactivity with 4-(4-nitrobenzyl) pyridine was also carried out. TCPO was more mutagenic in the preincubation test than in the plate test but NPO was the reverse. TCPO was more reactive than
any other epoxide tested in this study (Giri et al., 1989). The only negative results in Salmonella were reported by Dean et al. (1985) using TA1538, but these authors got positive results in both the E. coli WP2 and E. coli WP2 uvrA strains. Reports are also available on PO mutagenicity in other assays. PO can induce sex-linked lethal mutations in Drosophila (Schalet, 1954; Hardin et al., 1983). However, the dominant-lethal mutation study in rats and sperm-head morphology studies in mice could not detect any significant effects with PO (Hardin et al., 1983). PO also induced forward mutations in Chinese hamster ovary cells (Zamora et al., 1983). Cytogenetic assays The available information shows that both PO and T C P O can significantly induce chromosome aberrations (CA) and sister-chromatid exchanges (SCE) (Table 2). Two in vivo studies (Bootman et al., 1979; Lynch et al., 1984b) reported negative results in mice and monkeys. PO induced CA at a concentration below 2 / z g / m l in dividing human lymphocytes in culture (Bootman et al., 1979). However, no significant increase in micronucleated polychromatic erythrocytes was observed when PO was administered orally in mice given two doses of 100, 250 or 500 m g / k g body weight. But an increase in the incident of micronucleated cells in bone marrow was observed in mice given two intraperitoneal (i.p.) injections of 300 m g / k g body weight PO, 30 and 6 h prior to killing (Bootman et al., 1979). Dean and Hodson-Walker (1979) reported that PO can induce CA in an epithelial type cell line (RL 0 derived from rat liver. In flask cultures, PO produced a dose-related increase in chromatid damage with a striking increase in the incidence of chromatid gaps. An increased incidence of CA in cultured human lymphocytes was observed after 24-h treatment, but no significant increase in CA was observed after 48 h of treatment with T C P O (Norppa et al., 1981). A significant increase in SCE was also observed in lymphocytes treated with T C P O for 48 h. The induction of SCE was relatively less than with other epoxides like styrene oxide, epichlorohydrin and glycidol. The reasons that there were fewer CA and SCE in
cultured lymphocytes compared to other epoxides mentioned above could be TCPO's shorter halflife (Norppa et al., 1981). Population surveys of employees exposed for more than 20 years to alkylene oxides (including PO) showed a significant increase in CA (excluding gaps) in cultured lymphocytes when compared with controls (Thiess et al., 1981). The authors concluded that the populations were exposed to a number of products and it was difficult to predict that the risk was specific to one product. Exposure of cultured rat liver cells to PO in vitro showed a significant dose-related increase in chromatid aberrations at concentrations of 25-100 /zg/ml. It induced chromatid gaps in 56% of the cells at the higher dose (Dean et al., 1985). Similarly Gulati et al. (1989) reported that PO can induce significant increases in CA and SCE in Chinese hamster ovary cells in vitro with and without metabolic activation. However, no increases in CA or SCE were found in peripheral lymphocytes of male cynomolgus monkeys exposed to concentrations of 0, 237 and 717 mg P O / m 3 air, for 7 h per day, 5 days per week, for 2 years (Lynch et al., 1984b). Both NPO and T C P O were tested in in vivo genotoxicity assays in mice measuring CA and SCE. Trend tests for the evidence of dose-response effects were significant for both CA and SCE with both compounds. When the trends were compared between the two compounds, it appeared that the induction of SCE with NPO was more significant than with TCPO. No differences were observed between the two trends with CA (Giri et al., 1989). Interactions with nucleic acids A majority of the theories of carcinogenesis are based on the premise that most carcinogens are also mutagens. The literature indicates that PO can induce malignant tumours in rodents. Accordingly, to establish whether PO follows a mutagenic pathway in transforming normal cells to a neoplastic state, it is imperative to determine whether PO affects DNA. A number of publications are available regarding the interaction of PO and T C P O with nucleic acids. Walles (1974) reported that PO causes single-strand breaks in isolated calf thymus DNA, probably by alkylation
o
•~ =
~.~
~
" ~
~ "
~
~
•-~ ~-- "~ ~" ~ o o "~ ~ .~ ~ . ~ ~ "
~ ~
a o r',
Z
d G~
o •R
"
o o +
u
;
:
;+
I
+
:
;
;
:
;+
;
. 1 1 + : Z
o
i
z +
+ + + + + + + + + + + +
:
;
+
+
+ + ~ + :
Z
~.~
©
0
a
._~
~ ._.
z ~ ~
-I
Z
6~
~~
8 +~
of the phosphodiester bond. Alkylation of guanine detected by a simple fluorescence assay was reported by Hemminki (1979). Both PO and TCPO were tested and the order of reactivity was parallel to their mutagenic potency, TCPO being the more reactive alkylating agent. These epoxides alkylated deoxyguanosine faster than singlestranded DNA, at equal concentrations of guanine. The author concluded that the rate of alkylation depended on the molecular structure of the nucleophiles. Deoxyguanosine was alkylated faster than single-stranded DNA, which in turn was alkylated faster than double-stranded DNA. The molecular rigidity of macromolecules and particularly of double-stranded DNA could explain the difference in reaction rates. Hemminki et al. (1980) reported that both PO and TCPO reacting with deoxyguanosine produced a major adduct of 1,7- (or 1,9-) dialkylguanine. PO reacted with deoxyadenosine at N-6. PO, TCPO and other epoxides tend to react in a uniform way with nucleic acid bases (Hemminki et al., 1980). Djuric and Sinsheimer (1984a) reacted deoxycytidine with PO, glycidol, epichlorohydrin and TCPO which has varying alkylating rates. They found that although there were differences in the extent of alkylation at the nitrogen and oxygen sites for each epoxide, total reactivity with deoxy-
cytidine correlated to mutagenicity. They also reacted thymidine in methanol with PO and TCPO. A single product was detected and identified as 3-alkylthymidine with both compounds. Massspectrum analysis showed the products to be consistent with attachment at the least substituted carbon of the epoxide (Djuric and Sinsheimer, 1984b). In another study PO and TCPO were reacted with deoxyguanosine as well as deoxyadenosine and except for TCPO with DNA, and results correlated with mutagenicity and chemical reactivity (Djuric et al., 1986). Mutagenicity as determined by the plate incorporation method does not indicate a consistent correlation to epoxide deoxynucleoside reactivity. This is attributed to volatility and factors affecting the contact of mutagen with bacteria in the agar media in the plate vs. the preincubation test. They also indicated that TCPO more readily forms a ring-opened deoxyguanosine adduct, which could lead to a large increase in mutagenicity for TA100 over that for TA1535 because of the error-prone repair system in TA100 (Djuric et al., 1986). From the studies on interaction of PO and TCPO with DNA, it appears that both compounds can interact with DNA. Correlations between bacterial mutagenicity and alkylation rate
TABLE 2 C Y T O G E N E T I C ASSAYS I N D U C E D BY P R O P Y L E N E O X I D E A N D T R I C H L O R O P R O P Y L E N E O X I D E Chemical
Test system
E n d point
Effect
References
Propylene oxide
Mice (oral study) Mice (i.p. study) Rat liver cells ( R L 1 in vitro) C H O cells in vitro H u m a n lymphocytes in vitro H u m a n lymphocytes in vivo Monkey
MN MN CA
+ +
CA, SCE CA
+ +
Bootman et al. (1979) Bootman et al. (1979) Dean and Hodson-Walker (1979); D e a n et al. (1985) Gulati et al. (1989) Bootman et al. (1979)
CA
+
Thiess et al. (1981)
CA, SCE
-
Lynch et al. (1984b)
H u m a n lymphocytes in vitro Mice
CA, SCE
+
Norppa et al. (1981)
CA, SCE
+
Giri et al. (1989)
Trichloropropylene oxide
+ , positive effect; - , negative effect. CHO, Chinese hamster ovary cells; MN, micronucleus formation; CA, chromosome aberrations; SCE, sister-chromatid exchange.
with adduct formation in D N A were also observed in some experiments (Hemminki et al., 1980; Djuric and Sinsheimer, 1984a,b; Djuric et al., 1986). So it was quite evident that PO and T C P O follow a genetic pathway for transforming the normal cells to a neoplastic state in rodents. Discussion and conclusion
From the studies of genotoxicity and interactions with D N A by PO and TCPO, it appears that PO can act at the chromosomal and D N A levels. PO is mutagenic in bacteria, fungi and insects. PO is mutagenic in Salmonella strains TA100 and TA1535 as well as E. coli WP2. This indicates that PO can induce base-substitution mutations in strains of S. typhimurium and E. coli without metabolic activation. Negative results were reported in Salmonella strains TA1537, TA1538 and TA98 which detect frame-shift mutagens (McMahon et al., 1979; Pfeiffer and Dunkelberg, 1980; Dean et al., 1985). The mutagenicity of T C P O in the presence of liver $9 was reduced in S. typhimurium, S. cerevisiae and N. crassa (Callen and Ong, 1978). Bootman et al. (1979) could not detect decreased reversion in S. typhimurium or E. coli with $9, indicating that the $9 mixture had limited ability to promote conversion of PO to propylene glycol. Why do the in vivo experiments (dominant-lethal test in rats and mice and sperm-head abnormality in mice) show negative effects (Bootman et al., 1979; Hardin et al., 1983)? The negative results could be due to the inadequate exposure of the target cells to PO or the PO gets metabolized to a harmless derivative. These negative findings followed oral administration or inhalation studies. PO may be rapidly transformed to the propylene glycol in the liver and can also be eliminated via lung or other routes. Epoxide hydrolases which can catalyse this type of reaction are present in the hepatic microsomes (Huston, 1972). So the mutagenicity of PO depends on the availability of PO in the target cells. In the oral administration or inhalation studies PO did not reach the target cells (bone marrow or testis). This was also true in the study by Bootman et al. (1979) when oral administration of PO did not induce micronuclei (MN)
in bone marrow cells of mice but did induce MN when administered i.p. In the i.p. study, the tissue PO levels would be elevated prior to elimination via the lung or other routes. Cytogenetic studies of PO and TCPO were mostly for in vitro studies and indicate a positive effect on chromosomes. This supports the premise that both PO and T C P O were direct-acting mutagens. Only one study of populations exposed to PO and some other related epoxides showed an increased incidence of CA in human lymphocytes (Thiess et al., 1981). We have studied CA and SCE in bone marrow cells and DNA strand breaks (SB) in liver for both NPO and TCPO. The comparisons were made by a single-dose plate test and preincubation test in Salmonella strain TA100 with chemical reactivity (Girl et al., 1989). It has been shown that neither mutagenicity nor genotoxicity would correlate with reactivity for these two compounds. TCPO, the least genotoxic epoxide, is the most chemically reactive (Giri et al., 1989). Norppa et al. (1981) have also reported that the high alkylation rate of TCPO in comparison to their other epoxides contrasts with the low capacity of T C P O to induce CA and SCE results in cultured human lymphocytes. They propose a greater rate of detoxication and present evidence for a shorter half-life for T C P O to explain these differences. We postulated that TCPO with its much greater chemical reactivity and shorter half-life has a much lower concentration in the bone marrow over the period of time necessary to produce CA and SCE results. This may also be a factor in the case of PO. Other factors may play a role, such as alkylating ability. Although PO is chemically less reactive than TCPO, the few negative results in in vivo studies with PO are mainly due to its conversion to propylene glycol in the liver which reduces the availability of the amount of PO in the target cells necessary to express its clastogenicity. The negative findings of CA and SCE by Lynch et al. (1984b) in an inhalation study of PO in monkeys could be due to elimination via the lung and other routes. Negative results in one oral study in mice (Bootman et al., 1979) and the inhalation study by Lynch et al. (1984b) may be due to the non-availability of PO because of its rapid elimination, conversion by hepatic microso-
mal systems, and rapid elimination of its metabolites. PO and TCPO can interact with DNA in vivo and in vitro. Two reports on DNA strand breaks (Sina et al., 1983; Giri et al., 1990) induced by PO and TCPO showed positive effects. Recently we have studied the in vivo DNA SB in liver of mice, induced by NPO and TCPO. The half-life studies were also carried out for both compounds by 4-nitrobenzyl pyridine (NBP) reactions. The NBP absorbance at zero time adjusted for the dilution of TCPO is an indication of the relative alkylating ability of the epoxides. The absorbance together with the half-life values of both the compounds were NPO (0.236; 122 h) and TCPO (6.212; 2.7 h). We have carried out a comparative study of a genotoxic epoxide 1-naphthyl glycidyl ether (more genotoxic in bone marrow cells than the other 3 epoxides tested including TCPO) with TCPO for a much shorter period of time (1 h). There was a statistically significant increase in SB at 600 mg/kg of TCPO but not for 1-naphthyl glycidyl ether (NGE) and a significant difference in the trend tests ( P < 0.001) for the 2 compounds was observed for this time period (Giri et al., 1990). This could be due to the TCPO's greater strength as an alkylating agent but could only be demonstrated under conditions where TCPO's shorter half-life and high rate of detoxication would be compensated by high doses for a brief time to avoid a lethal effect. The order of genotoxicity in bone marrow cells was NGE > NPO > TCPO but in the SB study the reverse order (TCPO > NGE = NPO) was observed. This study emphasizes the importance of considering half-lives of directacting alkylating agents in the evaluation of their in vivo genotoxicities (Giri et al., 1989, 1990). Results on the genotoxicity and interaction with DNA induced by PO and TCPO show that these compounds can induce genetic damage in microorganisms and in vivo as well as in vitro in mammalian cells. This review updates and brings the possible genetic toxicology potential of these two compounds into perspective. Although there is not much information available on the genotoxic effects in populations directly exposed to these compounds, the available information on the carcinogenicity studies in rodents and the short-term genotoxicity results indicate that these
are potentially genotoxic compounds which can interact via DNA damage.
Acknowledgements The author is extremely grateful to Professor J.E. Sinsheimer, Professor of Medicinal Chemistry and Toxicology, University of Michigan, Ann Arbor, MI for his critical comments and valuable suggestions. Thanks are also due to Mrs. Reena Besaria and Mr. H.P. Paul for secretarial help.
References Berg, G.L. (1984) Farm Chemical Handbook, Meister, Willoughby, OH, 190 pp. Bootman, J., D.C. Lodge and H.E. Whalley (1979) Mutagenic activity of propylene oxide in bacterial and mammalian systems, Mutation Res., 67, 101-112. Callen, D.F., and T.M. Ong (1978) Effects of epoxide hydrase inhibitor 1,1,1-trichloropropylene-2,3-oxide on the genetic activity of aflatoxin B, metabolites in in vitro activation systems, Mutation Res., 49, 371-376. Cookson, M.J., P. Sims and P.L. Grover (1971) Mutagenicity of epoxides of polycyclic hydrocarbons correlates with carcinogenicity of the parent hydrocarbons, Nature New Biol., 234, 186-187. Dean, B.J., and G. Hodson-Walker (1979) An in vitro chromosome assay using cultured rat-liver cells, Mutation Res., 64, 239-337. Dean, B.J., T.M. Brooks, G. Hodson-Walker and D.H. Hutson (1985) Genetic toxicology testing of 41 industrial chemicals, Mutation Res., 153, 57-77. DeFlora, S. (1981) Study of 106 organic and inorganic compounds in Salmonella/microsome test, Carcinogenesis, 2, 283-298. Djuric, Z., and J.E. Sinsheimer (1984a) Reactivity of propylene oxides towards doexycytidine and identification of reaction products, Chem.-Biol. Interact., 50, 219-231. Djuric, Z., and J.E. Sinsheimer (1984b) Characterization and quantitation of 3-alkylthymidines from reaction of mutagenic propylene oxides with thymidine, Chem.-Biol. Interact., 52, 243-253. Djuric, Z., B.H. Hooberman, L.B. Rosman and J.E. Sinsheimer (1986) Reactivity of mutagenic propylene oxide with deoxynucleosides and DNA, Environ. Mutagen., 8, 369-383. Dunkelberg, H. (1982) Carcinogenicity of ethylene oxide and 1,2-propylene oxide upon intragastric administration to rats, Br. J. Cancer, 46, 924-933. Dunkelberg, H. (1981) Carcinogenic activity of ethylene oxide and its reaction products 2-chloroethanol, 2-bromoethanol, ethylene glycol and diethylene glycol. I. Carcinogenicity of ethylene oxide in comparison with 1,2-propylene oxide after subcutaneous administration in mice, Zbl. Bakt. Hyg. 1. Abt. Orig. B, 174, 383-404.
Ehrenberg, L., and S. Hussain (1981) Genetic toxicity of some important epoxides, Mutation Res., 86, 1-113. Garro, A.J., and R.A. Phillips (1980) Detection of mutageninduced lesions in isolated DNA by marker rescue of Bacillus subtilis phage ~105, Mutation Res., 73, 1-13. Giri, A.K. (1986) Mutagenic and genotoxic effects of 2,3,7,8tetrachlorodibenzo-p-dioxin - a review, Mutation Res., 168, 241-248. Giri, A.K., and S.S. Que Hee (1988) In vivo sister chromatid exchange induced by 1,2-dichloroethane on bone marrow cells of mice, Environ. Mol. Mutagen., 12, 331-334. Giri, A.K., T.S. Banerjee, G. Talukder and A. Sharma (1986a) Effects of dyes (Indigo carmine, Metanil yellow, Fast Green FCF) and nitrite in vivo on bone marrow cells of mice, Cancer Lett., 30, 315-320. Giri, A.K., G. Talukder and A. Sharma (1986b) Sister chromatid exchange induced by Metanil yellow and nitrite singly and in combination in vivo on mice, Cancer Lett., 31,299-303. Giri, A.K., E.A. Messerly and J.E. Sinsheimer (1989) Sister chromatid exchange and chromosome aberrations for four aliphatic epoxides in mice, Mutation Res., 224, 253-261. Giri, A.K., E.A. Messerly, P.K. Chakraborty, B.H. Hooberman and J.E. Sinsheimer (1990) DNA strand breaks in liver for four aliphatic epoxides in mice, Mutation Res., 242, 187-194. Gulati, D.K., K. Witt, B. Anderson, E. Zeiger and M.D. Shelby (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. III: Results with 27 chemicals, Environ. Mol. Mutagen., 13, 133-193. Hardin, B.D., R.L. Schuler, P.M. Meginnis, R.W. Niemeier and R.J. Smith (1983) Evaluation of propylene oxide for mutagenic activity in 3 in vivo test systems, Mutation Res., 117, 337-344. Hemminki, K. (1979) Fluorescence study of DNA alkylation by epoxides, Chem.-Biol. Interact., 28, 269-278. Hemminki, K., and K. Falck (1979) Correlation of mutagenicity and 4-(p-nitrobenzyl) pyridine alkylation by epoxides, Toxicol. Lett., 4, 103-106. Hemminki, K., J. Paasivirta, T. Kurkirinne and L. Virkki (1980) Alkylation products of DNA bases by simple epoxides, Chem.-Biol. Interact., 30, 259-270. Heslot, H. (1962) Etude quantitative de r6versions bioch6miques indicties chez la levure Schizosaccharomyces pombe par des radiations et des substances radiomim6tiques, Abh. Obsch. Akad. Wiss. Berlin, K1, Med., 1, 193-228. Hussain, S., and Osterman-Golkar (1984) Dose response relationships for mutations induced in E. coli by some model compounds, Hereditas, 101, 57-68. Huston, D.H. (1972) in: Foreign Compound Metabolism in Mammals, Vol. 2, The Chemical Society, London, 379 pp. International Agency for Research on Cancer (1985) Monograph on the Evaluation of the Carcinogenic Risk of Chemicals to Humans: Allyl compounds, aldehydes, epoxides and peroxides, Vol. 36, IARC, Lyon.
Kolmark, G., and N.H. Giles (1955) Comparative studies of mono-epoxides as inducer of reverse mutations in Neurospora, Genetics, 40, 890-902. Lynch, D.W., T.R. Lewis, W.J. Moorman, J.R. Burg, D.H. Groth, A. Khan, L.J. Acerman and B.Y. Cockrell (1984a) Carcinogenic and toxicologic effects of inhaled ethylene oxide and propylene oxide in F344 rats, Toxicol. Appl. Pharmacol., 76, 69-84. Lynch, D.W., T.R. Lewis, W.J. Moorman, J.R. Berg, D.K. Gulati, P. Kaur and P.S. Sabharwal (1984b) Sister chromatid exchange an chromosome aberrations in lymphocytes from monkeys exposed to ethylene oxide and propylene oxide by inhalation, Toxicol. Appl. Pharmacol., 76, 85-95. Manson, M. (1980) Epoxides - Is there a human health problem?, Br., J. Ind. Med., 37, 317-336. McMahon, R.E., J.C. Cline and C.Z. Thompson (1979) Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens, Cancer Res., 39, 682-693. Migliore, L., A.M. Rossi and N. Loprieno (1982) Mutagenic action of structurally-related alkene oxides on Schizosaccharomyces pombe: the influence, 'in vitro', of mouse liver metabolizing system, Mutation Res., 102, 425-437. Mukherjee, A., A.K. Giri, A. Sharma and G. Talukder (1988) Relative efficacy of short-term tests in detecting genotoxic effects of cadmium chloride in mice in vivo, Mutation Res., 206, 285-295. National Toxicology Program (1984) Toxicology and carcinogenesis studies of propylene oxide (CAS No. 75-65-9) in F344/N rats and B6C3F I mice (inhalation study) (NTP83-020; NIH Publ. No. 84-2523), US Department of Health and Human Services, Research Triangle Park, NC. Norppa, H., K. Hemminki, M. Sorsa and H. Vainio (1981) Effects of monosubstituted epoxides on chromosome aberrations and SCE in cultured human lymphocytes, Mutation Res., 91,243-250. Pfeiffer, E.H., and H. Dunkelberg (1980) Mutagenicity of ethylene oxide and propylene oxide and of the glycols and halohydrins formed from them during fumigation of food stuffs, Food Cosmet. Toxicol., 18, 115-118. Phillips, R.A., S.A. Zahler and A.J. Garro (1980) Detection of mutagen induced lesions in isolated DNA using a new Bacillus subtilis transformation-based assay, Mutation Res., 74, 267-281. Rosman, L.B., V. Gaddamidi and J.E. Sinsheimer (1987) Mutagenicity of aryl propylene and butylene oxides with Salmonella, Mutation Res., 189, 189-204. Roychaudhury, A., and A.K. Giri (1989) Effects of certain food dyes on chromosomes of Allium cepa, Mutation Res., 223, 313-319. Schalet, A. (1954) The mutagenic action of 1,2-propylene oxide and ethyl sulfate on mature sperm, Drosophila Inf. Serv., 28, 155. Sina, J.F., C.L. Bean, G.R. Dysart, V.I. Taylor and M.O. Bradley (1983) Evaluation of the alkaline elution/rat he-
patocyte assay as a predictor of carcinogenic/mutagenic potential, Mutation Res., 113, 357-391. Sinsheimer, J.E., E. Van Den Eeckhout, B.H. Hooberman and V.G. Beylin (1987) Detoxication of aliphatic epoxides by diol formation and glutathion conjugation, Chem.-Biol. Interact., 63, 75-90. Thiess, A.M., H. Schwegler, I. Fleig and W.G. Stocker (1981) Mutagenicity study of workers exposed to alkene oxides (ethylene oxide/propylene oxide) and derivatives, J. Occup. Med., 23, 343-347. U.S. Food and Drug Administration (1980) Synthetic Organic Chemicals, U.S. Production and Sales (1979) (USITC Publication 1099), U.S. Government Printing Office, Washington, DC, pp. 269-299.
Voogd, C.E., J.J. Van der Stel and J.J.J.A.A. Jacobs (1981) The mutagenic action of aliphatic epoxides, Mutation Res., 89, 269-282. Wade, D.R., S.C. Airy and J.E. Sinsheimer (1978) Mutagenicity of aliphatic epoxides, Mutation Res., 58, 217-223. Walles, S.A. (1974) The influence of some alkylating agents on the structure of DNA in vitro, Chem.-Biol. Interact., 9, 97-103. Zamora, P.O., T.M. Benson, A.K. Li and A.L. Brooks (1983) Evaluation of an exposure system using cells grown on collagen gels for detecting highly volatile mutagen in the C H O / H G P R T mutation assay, Environ. Mutagen., 5, 795-801.
1
MUTREV 07314
Genetic toxicology of propylene oxide and trichloropropylene oxide a review A.K. Giri Division of Toxicology, Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226001 (India) (Received 25 July 1991) (Revision received 17 October 1991) (Accepted 17 October 1991)
Keywords: Propylene oxide; Trichloropropylene oxide; Genotoxicity
Summary Aliphatic epoxides are a group of compounds extensively used in industry and as laboratory reagents, and can be produced as metabolic intermediates. An important aliphatic epoxide is propylene oxide, extensively used in the production of propylene glycol and polyols used in the manufacture of polyurethane polymers. The widespread human exposure to propylene oxide is of great health concern. In this review an attempt has been made to evaluate and update the genotoxic effects of propylene oxide and trichloropropylene oxide based on the available literature.
Aliphatic epoxides are widely used in industry and as laboratory reagents. Some of the aliphatic epoxides are highly toxic and some are also animal carcinogens. Because of the extensive use of epoxides, questions have been raised concerning their genotoxic potential (Manson, 1980; Ehrenberg and Hussain, 1981). An important aliphatic epoxide is propylene oxide (PO). PO is extensively used in the production of propylene glycol and polyols. Human populations are exposed mainly in industrial operations using PO. Polyols are used in the manufacture of polyurethane polymers. Propylene glycol, made
Correspondence: Dr. A.K. Giri, Scientist, Division of Toxicology, Central Drug Research Institute, Chattar Manzil Palace, Post Box 173, Lucknow 226001 (India). The C.D.R.I. communication number for this paper is 4877.
by the reaction of PO with water, is mainly used in the production of unsaturated polyester resins (IARC, 1985). PO is also used for sterilizing packaged food products in fumigation chambers (Berg, 1984). The U.S. Food and Drug Administration (1980) has approved the use of PO as a direct or indirect food additive, provided it does not exceed specified limits. There is sufficient evidence that PO is carinogenic in rodents. In an oral gavage study in rats, a dose-dependent increase was found in the incidence of local tumours, mainly squamous-cell carcinomas of the forestomach (Dunkelberg, 1982). In an inhalation study in mice and rats, PO produced haemangiomas and haemangiosarcomas of the nasal turbinates in mice and papillary adenomas of the nasal turbinates in rats (National Toxicology Program, 1984; Lynch et al., 1984a). PO also induced local fibrosarcomas after
subcutaneous administration to mice (Dunkelberg, 1981). During the past 25 years there has been an increasing number of reports on the genotoxicity and interaction with DNA of PO. Because of extensive occupational exposure to humans, much attention has been given to this compound. We have been testing the short-term genotoxic effects of different industrial chemicals including aliphatic epoxides and food additives (Giri, 1986; Giri et al., 1986a,b; Mukherjee et al., 1988; Giri and Que Hee, 1988; Roychaudhury and Giri, 1989; Giri et al., 1989, 1990). The mutagenic and genotoxic effects of PO and trichloropropylene oxide (TCPO) are reviewed.
Short-term mutagenicity assay The mutagenic effects of PO on microorganisms indicate that PO is a direct-acting mutagen which induces base-pair substitutions. It did not induce mutation in T2 bacteriophage (Cookson et al., 1971). Several authors have reported that PO is mutagenic in Salmonella typhimurium strains TA1535 and TA100 both with and without metabolic activation. It was also mutagenic in Bacillus subtilis and Escherichia coli (Table 1). Pfeiffer and Dunkelberg (1980) observed a dose-dependent increase in the number of revertants in Salmonella strains TA100 and TA1535 (PO 10-200/.~mole/plate) without metabolic activation. They found that TA100 was less sensitive than TA1535. No mutagenicity was observed in strains TA98 and TA1537. Similar results were reported by McMahon et al. (1979) and DeFlora (1981) with positive effects in TA1535 and TA100 but negative results in TA1537, TA1538 and TA98. Garro and Phillips (1980) described a mutagenesis assay which detects the induction of forward mutations in isolated DNA. The assay utilizes replicative form DNA of the temperate Bacillus subtilis phage q~105 and tests the ability of chemicals to induce lesions which inactivate phage genes involved in lysogen formation. PO (25-100 mM) was reacted with single-stranded qq05 DNA of B. subtilis in dimethylformamide buffer at 42°C for 30 min. The presence of mutagenic lesions in the treated DNA sample was detected by marker rescue with both the Hcr +
and H c r - recipient strains. The induction of clear plaque-forming units was significant with both strains. No consistent decrease in marker rescue activity was observed although there was a decrease in the frequency of clear plaque-forming units generated with the Hcr + host at the highest PO concentration (Garro and Phillips, 1980). Hemminki and Falck (1979) studied the mutagenicity and alkylation rate of reaction with 4(p-nitrobenzyl) pyridine for both PO and TCPO. They observed mutagenicity in Salmonella strain TA100 and E. coli WP2 uvrA for both compounds. A good correlation was observed in the mutagenicity and alkylation rate in the case of TCPO, but PO showed relatively less mutagenicity than its reactivity with 4-(p-nitrobenzyl) pyridine. These structurally related base-pair substitution mutagens do not require metabolic activation and Callen and Ong (1978) reported that the genetic activity of TCPO could be eliminated or reduced in the presence of the $9 fraction of liver homogenate. This can be explained by the rapid detoxication by glutathione and the rapid binding of TCPO to epoxide hydrolase and other proteins within the $9 fraction (Sinsheimer et al., 1987). This would result in a partial or complete absence of unbound TCPO able to penetrate the target cell and bind to DNA. The induction of mutation by PO in isolated transforming DNA of Bacillus subtilis was also reported by Phillips et al. (1980). They also studied the capacity of PO to inactivate transforming activity and disrupt gene linkage in treated DNAs. Although PO was a weak mutagen in this assay, it produced more extensive disruption of gene linkage at the concentration which produced comparable levels of inactivation of transforming activity. Rosman et al. (1987) also reported the mutagenic effects of several propylene oxide derivatives including 1-naphthyl propylene oxide (NPO) in Salmonella strains TA100 and TA1535 using both the preincubation and plate incorporation tests. We have also studied the mutagenicity of NPO and TCPO in preincubation and plate tests in Salmonella strain TA100 without $9. Chemical reactivity with 4-(4-nitrobenzyl) pyridine was also carried out. TCPO was more mutagenic in the preincubation test than in the plate test but NPO was the reverse. TCPO was more reactive than
any other epoxide tested in this study (Giri et al., 1989). The only negative results in Salmonella were reported by Dean et al. (1985) using TA1538, but these authors got positive results in both the E. coli WP2 and E. coli WP2 uvrA strains. Reports are also available on PO mutagenicity in other assays. PO can induce sex-linked lethal mutations in Drosophila (Schalet, 1954; Hardin et al., 1983). However, the dominant-lethal mutation study in rats and sperm-head morphology studies in mice could not detect any significant effects with PO (Hardin et al., 1983). PO also induced forward mutations in Chinese hamster ovary cells (Zamora et al., 1983). Cytogenetic assays The available information shows that both PO and T C P O can significantly induce chromosome aberrations (CA) and sister-chromatid exchanges (SCE) (Table 2). Two in vivo studies (Bootman et al., 1979; Lynch et al., 1984b) reported negative results in mice and monkeys. PO induced CA at a concentration below 2 / z g / m l in dividing human lymphocytes in culture (Bootman et al., 1979). However, no significant increase in micronucleated polychromatic erythrocytes was observed when PO was administered orally in mice given two doses of 100, 250 or 500 m g / k g body weight. But an increase in the incident of micronucleated cells in bone marrow was observed in mice given two intraperitoneal (i.p.) injections of 300 m g / k g body weight PO, 30 and 6 h prior to killing (Bootman et al., 1979). Dean and Hodson-Walker (1979) reported that PO can induce CA in an epithelial type cell line (RL 0 derived from rat liver. In flask cultures, PO produced a dose-related increase in chromatid damage with a striking increase in the incidence of chromatid gaps. An increased incidence of CA in cultured human lymphocytes was observed after 24-h treatment, but no significant increase in CA was observed after 48 h of treatment with T C P O (Norppa et al., 1981). A significant increase in SCE was also observed in lymphocytes treated with T C P O for 48 h. The induction of SCE was relatively less than with other epoxides like styrene oxide, epichlorohydrin and glycidol. The reasons that there were fewer CA and SCE in
cultured lymphocytes compared to other epoxides mentioned above could be TCPO's shorter halflife (Norppa et al., 1981). Population surveys of employees exposed for more than 20 years to alkylene oxides (including PO) showed a significant increase in CA (excluding gaps) in cultured lymphocytes when compared with controls (Thiess et al., 1981). The authors concluded that the populations were exposed to a number of products and it was difficult to predict that the risk was specific to one product. Exposure of cultured rat liver cells to PO in vitro showed a significant dose-related increase in chromatid aberrations at concentrations of 25-100 /zg/ml. It induced chromatid gaps in 56% of the cells at the higher dose (Dean et al., 1985). Similarly Gulati et al. (1989) reported that PO can induce significant increases in CA and SCE in Chinese hamster ovary cells in vitro with and without metabolic activation. However, no increases in CA or SCE were found in peripheral lymphocytes of male cynomolgus monkeys exposed to concentrations of 0, 237 and 717 mg P O / m 3 air, for 7 h per day, 5 days per week, for 2 years (Lynch et al., 1984b). Both NPO and T C P O were tested in in vivo genotoxicity assays in mice measuring CA and SCE. Trend tests for the evidence of dose-response effects were significant for both CA and SCE with both compounds. When the trends were compared between the two compounds, it appeared that the induction of SCE with NPO was more significant than with TCPO. No differences were observed between the two trends with CA (Giri et al., 1989). Interactions with nucleic acids A majority of the theories of carcinogenesis are based on the premise that most carcinogens are also mutagens. The literature indicates that PO can induce malignant tumours in rodents. Accordingly, to establish whether PO follows a mutagenic pathway in transforming normal cells to a neoplastic state, it is imperative to determine whether PO affects DNA. A number of publications are available regarding the interaction of PO and T C P O with nucleic acids. Walles (1974) reported that PO causes single-strand breaks in isolated calf thymus DNA, probably by alkylation
o
•~ =
~.~
~
" ~
~ "
~
~
•-~ ~-- "~ ~" ~ o o "~ ~ .~ ~ . ~ ~ "
~ ~
a o r',
Z
d G~
o •R
"
o o +
u
;
:
;+
I
+
:
;
;
:
;+
;
. 1 1 + : Z
o
i
z +
+ + + + + + + + + + + +
:
;
+
+
+ + ~ + :
Z
~.~
©
0
a
._~
~ ._.
z ~ ~
-I
Z
6~
~~
8 +~
of the phosphodiester bond. Alkylation of guanine detected by a simple fluorescence assay was reported by Hemminki (1979). Both PO and TCPO were tested and the order of reactivity was parallel to their mutagenic potency, TCPO being the more reactive alkylating agent. These epoxides alkylated deoxyguanosine faster than singlestranded DNA, at equal concentrations of guanine. The author concluded that the rate of alkylation depended on the molecular structure of the nucleophiles. Deoxyguanosine was alkylated faster than single-stranded DNA, which in turn was alkylated faster than double-stranded DNA. The molecular rigidity of macromolecules and particularly of double-stranded DNA could explain the difference in reaction rates. Hemminki et al. (1980) reported that both PO and TCPO reacting with deoxyguanosine produced a major adduct of 1,7- (or 1,9-) dialkylguanine. PO reacted with deoxyadenosine at N-6. PO, TCPO and other epoxides tend to react in a uniform way with nucleic acid bases (Hemminki et al., 1980). Djuric and Sinsheimer (1984a) reacted deoxycytidine with PO, glycidol, epichlorohydrin and TCPO which has varying alkylating rates. They found that although there were differences in the extent of alkylation at the nitrogen and oxygen sites for each epoxide, total reactivity with deoxy-
cytidine correlated to mutagenicity. They also reacted thymidine in methanol with PO and TCPO. A single product was detected and identified as 3-alkylthymidine with both compounds. Massspectrum analysis showed the products to be consistent with attachment at the least substituted carbon of the epoxide (Djuric and Sinsheimer, 1984b). In another study PO and TCPO were reacted with deoxyguanosine as well as deoxyadenosine and except for TCPO with DNA, and results correlated with mutagenicity and chemical reactivity (Djuric et al., 1986). Mutagenicity as determined by the plate incorporation method does not indicate a consistent correlation to epoxide deoxynucleoside reactivity. This is attributed to volatility and factors affecting the contact of mutagen with bacteria in the agar media in the plate vs. the preincubation test. They also indicated that TCPO more readily forms a ring-opened deoxyguanosine adduct, which could lead to a large increase in mutagenicity for TA100 over that for TA1535 because of the error-prone repair system in TA100 (Djuric et al., 1986). From the studies on interaction of PO and TCPO with DNA, it appears that both compounds can interact with DNA. Correlations between bacterial mutagenicity and alkylation rate
TABLE 2 C Y T O G E N E T I C ASSAYS I N D U C E D BY P R O P Y L E N E O X I D E A N D T R I C H L O R O P R O P Y L E N E O X I D E Chemical
Test system
E n d point
Effect
References
Propylene oxide
Mice (oral study) Mice (i.p. study) Rat liver cells ( R L 1 in vitro) C H O cells in vitro H u m a n lymphocytes in vitro H u m a n lymphocytes in vivo Monkey
MN MN CA
+ +
CA, SCE CA
+ +
Bootman et al. (1979) Bootman et al. (1979) Dean and Hodson-Walker (1979); D e a n et al. (1985) Gulati et al. (1989) Bootman et al. (1979)
CA
+
Thiess et al. (1981)
CA, SCE
-
Lynch et al. (1984b)
H u m a n lymphocytes in vitro Mice
CA, SCE
+
Norppa et al. (1981)
CA, SCE
+
Giri et al. (1989)
Trichloropropylene oxide
+ , positive effect; - , negative effect. CHO, Chinese hamster ovary cells; MN, micronucleus formation; CA, chromosome aberrations; SCE, sister-chromatid exchange.
with adduct formation in D N A were also observed in some experiments (Hemminki et al., 1980; Djuric and Sinsheimer, 1984a,b; Djuric et al., 1986). So it was quite evident that PO and T C P O follow a genetic pathway for transforming the normal cells to a neoplastic state in rodents. Discussion and conclusion
From the studies of genotoxicity and interactions with D N A by PO and TCPO, it appears that PO can act at the chromosomal and D N A levels. PO is mutagenic in bacteria, fungi and insects. PO is mutagenic in Salmonella strains TA100 and TA1535 as well as E. coli WP2. This indicates that PO can induce base-substitution mutations in strains of S. typhimurium and E. coli without metabolic activation. Negative results were reported in Salmonella strains TA1537, TA1538 and TA98 which detect frame-shift mutagens (McMahon et al., 1979; Pfeiffer and Dunkelberg, 1980; Dean et al., 1985). The mutagenicity of T C P O in the presence of liver $9 was reduced in S. typhimurium, S. cerevisiae and N. crassa (Callen and Ong, 1978). Bootman et al. (1979) could not detect decreased reversion in S. typhimurium or E. coli with $9, indicating that the $9 mixture had limited ability to promote conversion of PO to propylene glycol. Why do the in vivo experiments (dominant-lethal test in rats and mice and sperm-head abnormality in mice) show negative effects (Bootman et al., 1979; Hardin et al., 1983)? The negative results could be due to the inadequate exposure of the target cells to PO or the PO gets metabolized to a harmless derivative. These negative findings followed oral administration or inhalation studies. PO may be rapidly transformed to the propylene glycol in the liver and can also be eliminated via lung or other routes. Epoxide hydrolases which can catalyse this type of reaction are present in the hepatic microsomes (Huston, 1972). So the mutagenicity of PO depends on the availability of PO in the target cells. In the oral administration or inhalation studies PO did not reach the target cells (bone marrow or testis). This was also true in the study by Bootman et al. (1979) when oral administration of PO did not induce micronuclei (MN)
in bone marrow cells of mice but did induce MN when administered i.p. In the i.p. study, the tissue PO levels would be elevated prior to elimination via the lung or other routes. Cytogenetic studies of PO and TCPO were mostly for in vitro studies and indicate a positive effect on chromosomes. This supports the premise that both PO and T C P O were direct-acting mutagens. Only one study of populations exposed to PO and some other related epoxides showed an increased incidence of CA in human lymphocytes (Thiess et al., 1981). We have studied CA and SCE in bone marrow cells and DNA strand breaks (SB) in liver for both NPO and TCPO. The comparisons were made by a single-dose plate test and preincubation test in Salmonella strain TA100 with chemical reactivity (Girl et al., 1989). It has been shown that neither mutagenicity nor genotoxicity would correlate with reactivity for these two compounds. TCPO, the least genotoxic epoxide, is the most chemically reactive (Giri et al., 1989). Norppa et al. (1981) have also reported that the high alkylation rate of TCPO in comparison to their other epoxides contrasts with the low capacity of T C P O to induce CA and SCE results in cultured human lymphocytes. They propose a greater rate of detoxication and present evidence for a shorter half-life for T C P O to explain these differences. We postulated that TCPO with its much greater chemical reactivity and shorter half-life has a much lower concentration in the bone marrow over the period of time necessary to produce CA and SCE results. This may also be a factor in the case of PO. Other factors may play a role, such as alkylating ability. Although PO is chemically less reactive than TCPO, the few negative results in in vivo studies with PO are mainly due to its conversion to propylene glycol in the liver which reduces the availability of the amount of PO in the target cells necessary to express its clastogenicity. The negative findings of CA and SCE by Lynch et al. (1984b) in an inhalation study of PO in monkeys could be due to elimination via the lung and other routes. Negative results in one oral study in mice (Bootman et al., 1979) and the inhalation study by Lynch et al. (1984b) may be due to the non-availability of PO because of its rapid elimination, conversion by hepatic microso-
mal systems, and rapid elimination of its metabolites. PO and TCPO can interact with DNA in vivo and in vitro. Two reports on DNA strand breaks (Sina et al., 1983; Giri et al., 1990) induced by PO and TCPO showed positive effects. Recently we have studied the in vivo DNA SB in liver of mice, induced by NPO and TCPO. The half-life studies were also carried out for both compounds by 4-nitrobenzyl pyridine (NBP) reactions. The NBP absorbance at zero time adjusted for the dilution of TCPO is an indication of the relative alkylating ability of the epoxides. The absorbance together with the half-life values of both the compounds were NPO (0.236; 122 h) and TCPO (6.212; 2.7 h). We have carried out a comparative study of a genotoxic epoxide 1-naphthyl glycidyl ether (more genotoxic in bone marrow cells than the other 3 epoxides tested including TCPO) with TCPO for a much shorter period of time (1 h). There was a statistically significant increase in SB at 600 mg/kg of TCPO but not for 1-naphthyl glycidyl ether (NGE) and a significant difference in the trend tests ( P < 0.001) for the 2 compounds was observed for this time period (Giri et al., 1990). This could be due to the TCPO's greater strength as an alkylating agent but could only be demonstrated under conditions where TCPO's shorter half-life and high rate of detoxication would be compensated by high doses for a brief time to avoid a lethal effect. The order of genotoxicity in bone marrow cells was NGE > NPO > TCPO but in the SB study the reverse order (TCPO > NGE = NPO) was observed. This study emphasizes the importance of considering half-lives of directacting alkylating agents in the evaluation of their in vivo genotoxicities (Giri et al., 1989, 1990). Results on the genotoxicity and interaction with DNA induced by PO and TCPO show that these compounds can induce genetic damage in microorganisms and in vivo as well as in vitro in mammalian cells. This review updates and brings the possible genetic toxicology potential of these two compounds into perspective. Although there is not much information available on the genotoxic effects in populations directly exposed to these compounds, the available information on the carcinogenicity studies in rodents and the short-term genotoxicity results indicate that these
are potentially genotoxic compounds which can interact via DNA damage.
Acknowledgements The author is extremely grateful to Professor J.E. Sinsheimer, Professor of Medicinal Chemistry and Toxicology, University of Michigan, Ann Arbor, MI for his critical comments and valuable suggestions. Thanks are also due to Mrs. Reena Besaria and Mr. H.P. Paul for secretarial help.
References Berg, G.L. (1984) Farm Chemical Handbook, Meister, Willoughby, OH, 190 pp. Bootman, J., D.C. Lodge and H.E. Whalley (1979) Mutagenic activity of propylene oxide in bacterial and mammalian systems, Mutation Res., 67, 101-112. Callen, D.F., and T.M. Ong (1978) Effects of epoxide hydrase inhibitor 1,1,1-trichloropropylene-2,3-oxide on the genetic activity of aflatoxin B, metabolites in in vitro activation systems, Mutation Res., 49, 371-376. Cookson, M.J., P. Sims and P.L. Grover (1971) Mutagenicity of epoxides of polycyclic hydrocarbons correlates with carcinogenicity of the parent hydrocarbons, Nature New Biol., 234, 186-187. Dean, B.J., and G. Hodson-Walker (1979) An in vitro chromosome assay using cultured rat-liver cells, Mutation Res., 64, 239-337. Dean, B.J., T.M. Brooks, G. Hodson-Walker and D.H. Hutson (1985) Genetic toxicology testing of 41 industrial chemicals, Mutation Res., 153, 57-77. DeFlora, S. (1981) Study of 106 organic and inorganic compounds in Salmonella/microsome test, Carcinogenesis, 2, 283-298. Djuric, Z., and J.E. Sinsheimer (1984a) Reactivity of propylene oxides towards doexycytidine and identification of reaction products, Chem.-Biol. Interact., 50, 219-231. Djuric, Z., and J.E. Sinsheimer (1984b) Characterization and quantitation of 3-alkylthymidines from reaction of mutagenic propylene oxides with thymidine, Chem.-Biol. Interact., 52, 243-253. Djuric, Z., B.H. Hooberman, L.B. Rosman and J.E. Sinsheimer (1986) Reactivity of mutagenic propylene oxide with deoxynucleosides and DNA, Environ. Mutagen., 8, 369-383. Dunkelberg, H. (1982) Carcinogenicity of ethylene oxide and 1,2-propylene oxide upon intragastric administration to rats, Br. J. Cancer, 46, 924-933. Dunkelberg, H. (1981) Carcinogenic activity of ethylene oxide and its reaction products 2-chloroethanol, 2-bromoethanol, ethylene glycol and diethylene glycol. I. Carcinogenicity of ethylene oxide in comparison with 1,2-propylene oxide after subcutaneous administration in mice, Zbl. Bakt. Hyg. 1. Abt. Orig. B, 174, 383-404.
Ehrenberg, L., and S. Hussain (1981) Genetic toxicity of some important epoxides, Mutation Res., 86, 1-113. Garro, A.J., and R.A. Phillips (1980) Detection of mutageninduced lesions in isolated DNA by marker rescue of Bacillus subtilis phage ~105, Mutation Res., 73, 1-13. Giri, A.K. (1986) Mutagenic and genotoxic effects of 2,3,7,8tetrachlorodibenzo-p-dioxin - a review, Mutation Res., 168, 241-248. Giri, A.K., and S.S. Que Hee (1988) In vivo sister chromatid exchange induced by 1,2-dichloroethane on bone marrow cells of mice, Environ. Mol. Mutagen., 12, 331-334. Giri, A.K., T.S. Banerjee, G. Talukder and A. Sharma (1986a) Effects of dyes (Indigo carmine, Metanil yellow, Fast Green FCF) and nitrite in vivo on bone marrow cells of mice, Cancer Lett., 30, 315-320. Giri, A.K., G. Talukder and A. Sharma (1986b) Sister chromatid exchange induced by Metanil yellow and nitrite singly and in combination in vivo on mice, Cancer Lett., 31,299-303. Giri, A.K., E.A. Messerly and J.E. Sinsheimer (1989) Sister chromatid exchange and chromosome aberrations for four aliphatic epoxides in mice, Mutation Res., 224, 253-261. Giri, A.K., E.A. Messerly, P.K. Chakraborty, B.H. Hooberman and J.E. Sinsheimer (1990) DNA strand breaks in liver for four aliphatic epoxides in mice, Mutation Res., 242, 187-194. Gulati, D.K., K. Witt, B. Anderson, E. Zeiger and M.D. Shelby (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. III: Results with 27 chemicals, Environ. Mol. Mutagen., 13, 133-193. Hardin, B.D., R.L. Schuler, P.M. Meginnis, R.W. Niemeier and R.J. Smith (1983) Evaluation of propylene oxide for mutagenic activity in 3 in vivo test systems, Mutation Res., 117, 337-344. Hemminki, K. (1979) Fluorescence study of DNA alkylation by epoxides, Chem.-Biol. Interact., 28, 269-278. Hemminki, K., and K. Falck (1979) Correlation of mutagenicity and 4-(p-nitrobenzyl) pyridine alkylation by epoxides, Toxicol. Lett., 4, 103-106. Hemminki, K., J. Paasivirta, T. Kurkirinne and L. Virkki (1980) Alkylation products of DNA bases by simple epoxides, Chem.-Biol. Interact., 30, 259-270. Heslot, H. (1962) Etude quantitative de r6versions bioch6miques indicties chez la levure Schizosaccharomyces pombe par des radiations et des substances radiomim6tiques, Abh. Obsch. Akad. Wiss. Berlin, K1, Med., 1, 193-228. Hussain, S., and Osterman-Golkar (1984) Dose response relationships for mutations induced in E. coli by some model compounds, Hereditas, 101, 57-68. Huston, D.H. (1972) in: Foreign Compound Metabolism in Mammals, Vol. 2, The Chemical Society, London, 379 pp. International Agency for Research on Cancer (1985) Monograph on the Evaluation of the Carcinogenic Risk of Chemicals to Humans: Allyl compounds, aldehydes, epoxides and peroxides, Vol. 36, IARC, Lyon.
Kolmark, G., and N.H. Giles (1955) Comparative studies of mono-epoxides as inducer of reverse mutations in Neurospora, Genetics, 40, 890-902. Lynch, D.W., T.R. Lewis, W.J. Moorman, J.R. Burg, D.H. Groth, A. Khan, L.J. Acerman and B.Y. Cockrell (1984a) Carcinogenic and toxicologic effects of inhaled ethylene oxide and propylene oxide in F344 rats, Toxicol. Appl. Pharmacol., 76, 69-84. Lynch, D.W., T.R. Lewis, W.J. Moorman, J.R. Berg, D.K. Gulati, P. Kaur and P.S. Sabharwal (1984b) Sister chromatid exchange an chromosome aberrations in lymphocytes from monkeys exposed to ethylene oxide and propylene oxide by inhalation, Toxicol. Appl. Pharmacol., 76, 85-95. Manson, M. (1980) Epoxides - Is there a human health problem?, Br., J. Ind. Med., 37, 317-336. McMahon, R.E., J.C. Cline and C.Z. Thompson (1979) Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens, Cancer Res., 39, 682-693. Migliore, L., A.M. Rossi and N. Loprieno (1982) Mutagenic action of structurally-related alkene oxides on Schizosaccharomyces pombe: the influence, 'in vitro', of mouse liver metabolizing system, Mutation Res., 102, 425-437. Mukherjee, A., A.K. Giri, A. Sharma and G. Talukder (1988) Relative efficacy of short-term tests in detecting genotoxic effects of cadmium chloride in mice in vivo, Mutation Res., 206, 285-295. National Toxicology Program (1984) Toxicology and carcinogenesis studies of propylene oxide (CAS No. 75-65-9) in F344/N rats and B6C3F I mice (inhalation study) (NTP83-020; NIH Publ. No. 84-2523), US Department of Health and Human Services, Research Triangle Park, NC. Norppa, H., K. Hemminki, M. Sorsa and H. Vainio (1981) Effects of monosubstituted epoxides on chromosome aberrations and SCE in cultured human lymphocytes, Mutation Res., 91,243-250. Pfeiffer, E.H., and H. Dunkelberg (1980) Mutagenicity of ethylene oxide and propylene oxide and of the glycols and halohydrins formed from them during fumigation of food stuffs, Food Cosmet. Toxicol., 18, 115-118. Phillips, R.A., S.A. Zahler and A.J. Garro (1980) Detection of mutagen induced lesions in isolated DNA using a new Bacillus subtilis transformation-based assay, Mutation Res., 74, 267-281. Rosman, L.B., V. Gaddamidi and J.E. Sinsheimer (1987) Mutagenicity of aryl propylene and butylene oxides with Salmonella, Mutation Res., 189, 189-204. Roychaudhury, A., and A.K. Giri (1989) Effects of certain food dyes on chromosomes of Allium cepa, Mutation Res., 223, 313-319. Schalet, A. (1954) The mutagenic action of 1,2-propylene oxide and ethyl sulfate on mature sperm, Drosophila Inf. Serv., 28, 155. Sina, J.F., C.L. Bean, G.R. Dysart, V.I. Taylor and M.O. Bradley (1983) Evaluation of the alkaline elution/rat he-
patocyte assay as a predictor of carcinogenic/mutagenic potential, Mutation Res., 113, 357-391. Sinsheimer, J.E., E. Van Den Eeckhout, B.H. Hooberman and V.G. Beylin (1987) Detoxication of aliphatic epoxides by diol formation and glutathion conjugation, Chem.-Biol. Interact., 63, 75-90. Thiess, A.M., H. Schwegler, I. Fleig and W.G. Stocker (1981) Mutagenicity study of workers exposed to alkene oxides (ethylene oxide/propylene oxide) and derivatives, J. Occup. Med., 23, 343-347. U.S. Food and Drug Administration (1980) Synthetic Organic Chemicals, U.S. Production and Sales (1979) (USITC Publication 1099), U.S. Government Printing Office, Washington, DC, pp. 269-299.
Voogd, C.E., J.J. Van der Stel and J.J.J.A.A. Jacobs (1981) The mutagenic action of aliphatic epoxides, Mutation Res., 89, 269-282. Wade, D.R., S.C. Airy and J.E. Sinsheimer (1978) Mutagenicity of aliphatic epoxides, Mutation Res., 58, 217-223. Walles, S.A. (1974) The influence of some alkylating agents on the structure of DNA in vitro, Chem.-Biol. Interact., 9, 97-103. Zamora, P.O., T.M. Benson, A.K. Li and A.L. Brooks (1983) Evaluation of an exposure system using cells grown on collagen gels for detecting highly volatile mutagen in the C H O / H G P R T mutation assay, Environ. Mutagen., 5, 795-801.
