Chem. Res. Toxicol. 1990,3,301-306
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Identification of Organic N-Chloramines in Vitro in Stomach Fluid from the Rat after Chlorination Frank E. Scully, Jr.,**t Kathryn E. Mazina,t H. Paul Ringhand,* Edward K. Chess,g James A. Campbell,$ and J. Donald Johnson11 Department of Chemical Sciences, Old Dominion University, Norfolk, Virginia 23529-0126, Health Effects Research Laboratory, US.Environmental Protection Agency, Cincinnati, Ohio 45268, Battelle Pacific Northwest Laboratories, P.O. Box 999, Richland, Washington 99352, and Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27514 Received August 14, 1989
Amino acids were identified and quantified in the stomach fluid recovered from animals fasted 8, 24, and 48 h. After in vitro chlorination of stomach fluid, three chloramino acids, Nchloroglycine, either N-chloroleucine or N-chloroisoleucine, and N-chlorophenylalanine, were identified by GC/MS analysis of dansylated methyl ester derivatives. Formation of Nchlorovaline and N-chloroserine was suggested by their HPLC retention times, but their identification could not be confirmed by GC/MS data.
Introduction In the United States the average person drinks between 1 and 2 L of water each day which usually contains a disinfectant such as aqueous chlorine. Over the past 15 years there has been considerable interest in the possible health effects associated with drinking chlorinated water. Most of this interest has focused on ingestion of trace quantities of byproducts of the chlorination of naturally occurring organic substances (humic substances) present in surface and ground waters. Little attention has been given to the potential health effects of the reactions of potent oxidizing and chlorinating disinfectants in the organic-rich media of saliva and stomach fluid. Toxicological studies of the effects of ingestion of aqueous chlorine are few. Abdel-Rahman et al. ( 1 ) has found that blood glutathione levels in Sprague-Dawleyrats decrease significantly as early as 30 min after acute exposure to aqueous chlorine solutions with concentrations as low as 10 and 40 mg/L (1.4 X lo4 and 5.6 X M). Glutathione is released from cells in response to xenobiotics and the activity of oxidants such as hydrogen peroxide (2). Excessive reduction of glutathione levels in red blood cells can lead to cell lysis. Meier et al. (3)found that oral administration of aqueous chlorine (pH 8.5) to B6C3F1 mice at levels of 4 and 8 mg/kg body weight per day for 5 weeks induced sperm-head abnormalities. The stomach is a dynamic system. The sight, smell, or taste of food or drink can induce the secretion of copious amounts of gastric fluid which contains hydrochloric acid (which lowers the pH of the stomach contents), salts, and enzymes needed to digest food. Consequently, the composition of the resulting stomach fluid can vary with the kinds of food ingested and with the amount of time following ingestion of food. Because aqueous chlorine is a highly reactive oxidant and chlorinating agent, any health effects associated with it8 ingestion are probably not due to the oxidant itself, but 'Old Dominion University. U.S. Environmental Protection Agency. Battelle Pacific Northwest Laboratories. 1 University of North Carolina.
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0893-228x/90/2703-0301$02.50/0
to the products of its reactions with organic or inorganic compounds present in saliva or stomach fluid. Furthermore, since the ch1orine:carbon reaction ratio is much smaller than that found in a drinking water treatment system, the products of the reactions of aqueous chlorine and chloramine in saliva and stomach fluid are not likely to be as extensively chlorinated or oxidized as previously identified byproducts of drinking water chlorination like chloroform and trichloroacetic acid. Because the fate of ingested chlorine in the body is not understood, there is a need to identify the reactions aqueous chlorine can undergo in the stomach that might account for observed toxicological effects. Since aqueous chlorine reacts rapidly with amino nitrogen compounds ( 4 , 5 )to form chloramines, it was hypothesized that, upon ingestion, chlorinated water would react rapidly with amino nitrogen compounds in stomach fluid and that N-chloramino byproducts would be primary products of the chlorination of organic compounds in the body. Recently, it was shown that N-chloroglycine and Nchloropiperidine can be formed in vivo in the stomach of laboratory rats after successive administration of the parent amine and aqueous chlorine (6). In this paper it will be shown that organic N-chloramines can be formed from the reaction of aqueous chlorine with amino acids naturally present in the stomach fluid of rats. Part of the impetus for the research reported here has been the previous observation that at least one organic N-chloramine, N-chloropiperidine, is weakly mutagenic by a modification of the Salmonella reverse mutation assay (Ames test) (7), is cytotoxic (8),and induces chromosomal aberrations in mammalian cells, the frequency of which is proportional to the concentration of the chloramine (9). A method for the derivatization and analysis of organic N-chloramines in dilute aqueous solution has been described (IO). Solutions containing N-chloramines can be reacted with 5-(dimethylamino)naphthalene-l-sulfinicacid to produce highly fluorescent sulfonamide derivatives (dansyl derivatives) which can be analyzed by high-performance liquid chromatography (HPLC). This technique was used to detect the formation in vivo of N-chloroglycine in the stomach fluid of animals administered chlorinated 0 1990 American Chemical
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302 Chem. Res. Toxicol., Vol. 3, No. 4, 1990
water (6). In the present study this derivatization method is used to detect the formation in vitro of several Nchloramino acids i n chlorinated stomach fluid by using
GC/MS.
Materials and Methods General Procedures. Preparation and standardization of aqueous chlorine solutions have been described previously (6). The HPLC equipment used in this study has been described elsewhere (10). Amino acids present in stomach fluid were identified and quantified by the method of Jones et al. (11). Dansyl amino acid standards were obtained from Sigma Chemical Co. Gas chromatographic/mass spectrometric analyses were performed on a Hewlett-Packard Model 5985A GC/MS using on-column injection and electron impact ionization (70 eV). A 30 m X 0.25 mm i.d. SE-54 column (J&W Scientific, Folsom, CA) with a 0.25-pm film thickness was temperature programmed from 50 to 300 "C at 15 OC/min. The spectrometer was tuned daily with perfluorotributylamine and was typically operated by 3canning from 50 to 450 amu at 1s/scan with a source temperature of 200 "C. The molecular weights of the standards were confirmed by analysis using methane chemical ionization. Operating conditions were the same as for El with the following exceptions: scan range @e480 amu), electron energy (230 eV), emission current (300 A),and ion source housing pressure (2 X lo-' Torr). Selected samples and standards were trimethylsilylated prior to GC/MS analysis by reaction with bis(trimethylsily1)trifluoroacetamide (BSTFA)/ 1% trimethylchlorosilane (TMCS) purchased from Pierce Chemical Co. (Rockford, IL). Standard reference samples of dansyl amino acid methyl esters were prepared either by methylation of the dansyl amino acid with ethereal diazomethane (12) or by reaction overnight of 0.5 mmol of the amino acid methyl ester with 0.5 mmol of dansyl chloride in 4 mL of acetonitrile containing 100 pL of 10 N NaOH. After concentration of the reaction solution on a rotary evaporator, the products were extracted into ether, the ether layer was dried over anhydrous sodium sulfate, and the compounds allowed to crystallize overnight. Their purity was checked by HPLC and by GC (isothermal at 300 OC, 30 m X 0.32 mm i.d. SPB-5 column with 0.25-rm film thickness, Supelco, Inc.). HPLC of samples prepared from dansyl chloride revealed the presence of trace quantities (
