Haloacetonitriles: metabolism, genotoxicity, and tumor-initiating activity.

Haloacetonitriles (HAN) are drinking water contaminants produced during chlorine disinfection. This paper evaluates metabolism, genotoxicity, and tumor-initiating activity of these chemicals. The alkylating potential of the HAN to react with the electrophile-trapping agent, 4-(p-nitrobenzyl)pyridine, followed the order dibromoacetonitrile (DBAN) greater than bromochloroacetonitrile (BCAN) greater than chloroacetonitrile (CAN) greater than dichloroacetonitrile (DCAN) greater than trichloroacetonitrile (TCAN). When administered orally to rats, the HAN were metabolized to cyanide and excreted in the urine as thiocyanate. The extent of thiocyanate excretion was CAN greater than BCAN greater than DCAN greater than DBAN much greater than TCAN. Haloacetonitriles inhibited in vitro microsomal dimethylnitrosamine demethylase (DMN-DM) activity. The most potent inhibitors were DBAN and BCAN, with Ki = 3-4 X 10(-5) M; the next potent were DCAN and TCAN, with Ki = 2 X 10(-4) M; and the least potent inhibitor was CAN, with Ki = 9 X 10(-2) M. When administered orally, TCAN, but not DBAN, inhibited hepatic DMN-DM activity. The HAN produced DNA strand breaks in cultured human lymphoblastic (CCRF-CEM) cells. TCAN was the most potent DNA strand breaker, and BCAN greater than DBAN greater than DCAN greater than CAN, which was only marginally active. DCAN reacted with polyadenylic acid and DNA to form adducts in a cell-free system; however, the oral administration of DBAN or DCAN to rats did not result in detectable adduct formation in liver DNA. None of the HAN initiated gamma-glutamyltranspeptidase (GGT) foci when assayed for tumor-initiating activity in rat liver foci bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
Chlorine is routinely used as a municipal drinking water disinfectant. However, chlorination of drinking water produces a variety of chlorinated organic compounds (1), including trihalomethanes and halogenated acetonitriles (HAN). Dichloroacetonitrile (DCAN), the most prevalent of the haloacetonitriles, has been routinely detected in drnking water at 0.3-8.1 ppb, which is about 10% of the molar concentration for trihalomethanes (2). DCAN has been reported (3) to be mutagenic in Salmonella typhimurium, so that exposure of humans to haloacetonitriles via drinking water might present a genotoxic and carcinogenic hazard. Thus the metabolism, genotoxicity, and tumor-initiating activity of HAN were evaluated.

Alkylation Potential
The HAN  (NBP) to form a colored product (4). Linear dose-response curves were obtained when absorbance at 570 nm of the colored product was plotted against five different concentrations of HAN. The extent of reaction was calculated as the slope of absorbance at 570 nm vs. the pi M test chemical curve after a linear-regression fit of the data. The results were standardized to chloroacetonitrile (CAN) equal to one and are presented in Table 1. CAN was the most active among the chlorinated HAN, and each additional chlorine substitution reduced reactivity by a factor of approximately ten. Substitution of chlorine by bromine increased reactivity, and DBAN was 100 times more reactive than DCAN. These results were similar to those obtained and cited by Epstein et al. (5) with alkyl halides: RCH2CH2X > RCH2CHX2 > RCH2CX3 and iodide > bromide > chloride.

Urinary Excretion of HAN as Thiocyanate
The thiocyanate content of 24-hr urine samples was determined after oral administration of 0.75 mmole/kg of HAN (6). The percentage of the dose excreted in the urine as thiocyanate was standardized to CAN equal to 'Alkylation potential as determined by binding to 4-(p-nitrobenzyl)pyridine (4).
b Urinary excretion as thiocyanate in 24 hr (6). one (Table 1). CAN and bromochloroacetonitrile (BCAN) were excreted to a greater extent than DCAN and dibromoacetonitrile (DBAN), which were excreted to a greater extent than trichloroacetonitrile (TCAN). Figure 1 depicts a proposed pathway for the metabolism of HAN to cyanide, which is further metabolized by rhodanese to thiocyanate. Hydroxylacetonitriles were proposed to form either by direct displacement of halide ion by an hydroxyl group or by oxidation of a hydrogen atom through the action of mixed-function oxidase. The carbonyl group is formed from the hydroxylacetonitrile by the elimination of cyanide ion or by the release of halide ion, forming cyanoformaldehyde or cyanoformyl halide. Cyanide ion could then be formed by the release of carbon monoxide from cyanoformaldehyde or by release of carbon dioxide from cyanoformic acid, which is formed by the hydrolysis of cyanoformyl halide. A similar metabolic pathway for the release of cyanide from nitriles has been proposed by Silver et al. (7).

Inhibition of Dimethylnitrosamine Demethylase
The HAN inhibited rat microsomal dimethylnitrosamine-demethylase (DMN-DM) activity in vitro (6). The dose-response relationship of inhibition by HAN of DMN-DM activity is presented in Figure 2.

DNA Strand Breaks in Human Lymphoblastic Cells
The HAN were incubated with CCRF-CEM cells, a human lymphoblastic line of T-cell origin, and the induction of DNA strand breaks was assayed by the alkaline unwinding procedure (4). All five HANs were able to induce DNA strand breaks. The extent of DNA strand breaks induced by the five HAN after 1 hr of exposure was standardized to CAN = 1.00 and is presented in Table 1. The HAN exhibited a range of potency, with TCAN the most potent, causing twice as many breaks as the genotoxic methylating agents methylmethanesulfonate and methylnitrosourea (4). The two bromine-containing HAN were cytotoxic to the CCRE-CEM cell, producing a 13 to 60% rate of cell  Peak IV from DCAN-bound Poly A and a minor peak (peak II; Fig. 3C). Strong acid hydrolysis of DCANbound DNA resulted in the disappearance of peak II with the occurrence of peaks III and IV in the region similar to peaks III and IV of the elution profile of DCAN-bound Poly A (Fig. 3D). Thus, the adduct present in peak IV of the elution proffle of DCAN-bound Poly A appears also to be formned when DCAN binds to DNA. In conclusion, DCAN has been shown to bind directly to DNA with the formation of an adduct to adenosine and possibly other sites in the DNA. However, oral adininistration of ['4C]-DBAN or ["C]-DCAN did not result in any detectable adduct formation in rat liver (results not shown).

Cancer Initiation Bioassay
An initiation-promotion bioassay in rat liver, the rat liver foci bioassay, is being developed for determining the tumor-initiating activity of chemical carcinogens (8). Briefly, the test substance is administered to partially hepatectomized rats followed 1 week later by the admin-   (9) aThe test substance was administered to Fischer-344 rats 24 hr after a 2/3 partial hepatectomy. One week after the operation, the animals started to receive 500 ppm sodium phenobarbital for a total of 8 weeks. One week after the cessation of treatment with phenobarbital, the animals were sacrificed and the liver was scored for the occurrence of GGT foci. bMean ± SE. The number of animals is presented in parentheses.
istration of 500 ppm sodium phenobarbital in the drinking water for a total of8 weeks. The partial hepatectomy is used to increase the sensitivity of the assay by inducting DNA replication during the binding of the test substance to DNA (9)(10)(11). The sodium phenobarbital is used to promote the appearance of preneoplastic and neoplastic lesions (12,13). The putative preneoplastic lesion, foci of hepatocytes containing y-glutamyltranspeptidase (GGT) activity, is used to indicate the occurrence of carcinogenesis initiation. Hepatocytes of rat liver lack GGT activity, whereas tumors contain GGT activity, especially when phenobarbital is used as a promoter (12,13). The initiation of hepatocarcinogenesis is believed to result in the occurrence of the morphologically altered cells that progress through clonal expansion into foci of altered hepatocytes (12,14). These foci of altered hepatocytes can, after further alteration (rare events), progress to neoplastic lesions. GGT foci are one type of altered foci used to indicate the occurrence of cancer initiation (12,14). Orally administered HAN were tested in rat liver foci bioassay for tumor-initiating activity ( Table 3). None of the four HAN induced GGT foci in rat liver. Diethylnitrosamine, which was used as a positive control, did induce GGT foci. Thus, the HAN appear to lack tumorinitiating activity in rat liver.

Conclusions
The HAN have been found in chlorinated drinking water (3,15) and have been shown to be formed in the stomach of rats after gavage administration of an aqueous solution of chlorine (16). Bromide in an aqueous solution of chlorine is oxidized to hypobromite (17). Thus, chlorination of drinking water that contains bromide could result in the formation of chlorineand bromine-containing HAN. The presence of HAN in drinking water or the production of HAN in the gastrointestinal tract after consumption of drinking water containing residue chlorine might pose a human health hazard.
Simmon et al. (3) have reported that DCAN is mutagenic in the Ames Salmonella assay. The HAN also induce sister-chromatid exchange in Chinese hamster ovary (CHO) cells (18). In this paper, we report that the HAN possessed direct-acting alkylating activity, inhibited DMN-DM activity in vitro, bound in vitro to polynucleotides including DNA, induced DNA strand breaks in cultured human lymphoblastic cells of T-cell origin, were excreted as thiocyanates in the urine, and did not exhibit tumor-initiating activity in the rat liver foci bioassay. The results of these chemical and biological evalutions of the HAN are summarized in Table 1.
No correlation was observed between their alkylation potential measured by their binding to NBP and their ability to produce DNA strand breaks in cultured cells or their ability to inhibit microsomal DMN-DM activity.
A nucleophilic reagent can react with HAN either by displacing a halogen atom or by attacking the carbon atom of the cyano group that has a partial positive charge resulting from the resonance: NBP probably reacts with HAN through displacement of halogen, a conjecture derived from the similarity of results between HAN and alkyl halides (5). Under similar conditions, amyl cyanide did not react with NBP (5). An electron-withdrawing substituent on the a-carbon increases the reaction rate of nucleophilic addition toward the cyano group (18). Thus, TCAN would be the most reactive among the HAN toward nucleophilic addition on the cyano group in contrast to the nucleophilic substitution of halogen. The contrasting results between the binding of HAN to NBP and their ability to cause DNA strand breaks or to inhibit DMN-DM activity could be the result of NBP reaction occurring by displacement of halogen, whereas the other effects occur both by displacement of halogen and by attack on the cyano group.
The HAN are absorbed systemically and are converted to toxic metabolites as demonstrated by their excretion as thiocyanates and their inhibition of DMN-DM activity in rat liver. However, the HAN failed to produce any detectable amounts of DNA adducts in rat liver following oral administration, failed to initiate GGT foci in the rat liver foci bioassay, and failed to induce micronuclei in polychromatic erythrocytes of bone marrow of CD-1 mice (19). This inability to demonstrate a systemic activity for HAN could be caused by the failure of these compounds to reach the target organ and/or to their rapid in vivo detoxification. The tumor-initiating activity of HAN in mouse skin when applied topically indicates that a carcinogenic hazard may exist at the site of application. Thus, if HAN which are present in drinking water or which are formed in the gastrointestinal tract after consumption of drinking water containing a residue of chlorine represent a carcinogenic hazard, the site of the hazard would most likely be limited to the gastrointestinal tract. Further investigations are required to determine whether the HAN are gastrointestinal carcinogens.