Carl C. Smith

Residue organic mixtures from drinking water show in vitro mutagenic and transforming activity.

Indications of possible health effects of residue organics in drinking water have been sought using short-term tests of mutagenic and transforming activity. Ten percent or less of the total organic material in drinking water has been identified; the remainder is believed to include thousands of unknown nonvolatile compounds. Residual organics were concentrated from drinking water from representative U.S. cities by reverse osmosis followed by liquid-liquid extraction [yielding the reverse osmosis concentrate-organic extract (ROC-OE) fraction] and sorption-desorption on XAD-2 resin. Samples of these residue organics were provided by the Environmental Protection Agency for bioassay. They were examined for mutagenic activity by using Salmonella tester strains (primarily TA98 and TA100) and for transforming activity by using mouse fibroblasts (BALB/3T3 clone 1-13). City-specific patterns of dose-dependent bacterial mutagenesis and of bacterial toxicity were observed for these samples and for subfractions generated by sequential extractions with hexane, ethyl ether, and acetone. Mutagenic effects were essentially independent of a microsome activation system prepared from liver of Aroclor 1254-induced rats. On the basis of strain-specific effects in mutagenesis and differential distributions of mutagenic activity during liquid-liquid extraction, at least some of the active compounds are thought to be acidic, frameshift mutagens. The ROC-OE fraction of a New Orleans sample transformed BALB/3T3 cells in replicate experiments. By comparison with the bacterial mutagenesis data, cell transformation is a relatively sensitive method for detecting possible mutagenic and carcinogenic activity in this sample. The appropriateness of these systems for the assay of complex mixtures and the degree to which reverse osmosis concentrates contain the unaltered organic compounds in the original samples are discussed.

Non-mutagenicity for salmonella of the chlorinated hydrocarbons aroclor 1254, 1,2,4-trichlorobenzene, mirex and kepone

A polychlorinated biphenyl mixture, Aroclor 1254, two commercial grade insecticides, mirex and kepone, and a pesticide breakdown product, 1,2,4-trichlorobenzene were evaluated for mutagenicity and hepatic enzyme induction potential in the Salmonella/microsomal assay. None was found to revert strains TA1535, TA1537, TA98 or TA100 when tested with or without metabolic activation. Liver microsomal extracts (S9) from rats induced with 1,2,4-trichlorobenzene were shown to differ from S9 of either control or Aroclor 1254-induced rats in the capacity to activate 2-aminoanthracene mutagenesis.

Mutagenic Studies of Folic Acid Antagonists

Compounds that compete with folic acid (folic acid antagonists [FAAs]) become limited in their usefulness in the treatment of leukemia, malaria, and bacterial infections by the rapid development of resistance. Assays of the plasma levels of certain of these FAAs led to the observation, in about 25% of the determinations, that a higher density of growth of Streptococcus faecium var. durans (ATCC 8043) was obtained at an FAA concentration just below the completely inhibitory level than at one-half this concentration. This and other considerations suggested that FAAs may act not only as selective agents for resistant organisms but also as mutagens. Seven FAAs including amethopterin, pyrimethamine, trimethoprim, chlorguanide triazine, an experimental quinazoline, WR-158,122, and two experimental triazines, WR-99,210 and WR-38,839, were tested for mutagenicity in the Salmonella reversion assay developed by Ames et al. (1975). All were found to be negative for strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without microsomal activation. These compounds were then tested as mutagens for three traits in the folic acid-requiring S. faecium. FAAs were shown to cause mutations to folic acid independence, rifampin resistance, and FAA resistance. It is postulated that the FAAs induce mutations by causing thymine deprivation in the folic acid-requiring host. Images

Blood Pressure of the Normal Rhesus Monkey

Summary Arterial blood pressures have been determined in unanesthetized monkeys by 2 indirect methods. Using a small “newborn” cuff (cloth cuff) on the upper arm and auscultation of the sounds in the brachial artery, mean blood pressure in 14 normal monkeys was found to range from 137/112 to 188/152 mm Hg with a group average of 159/127. With a cuff consisting of a rigid metal shell enclosing a rubber sleeve (metal cuff), mean pressures ranged from 103 78 to 132/101, the group average being 118 90 mm. In 27 splenectomized, but otherwise normal, monkeys the blood pressures were slightly but not significantly lower. Metal cuff pressure readings were found to be consistently less variable than corresponding cloth cuff values, the respective average standard deviations in normotensive animals being 7 and 12 mm. Blood pressures in untreated animals estimated by either indirect method were relatively stable; in no instance did mean systolic or diastolic pressure during a 12-month period vary by as much as 10 mm Hg from the mean values obtained during an initial 2-month observation period. Simultaneous comparisons of indirect (cloth or metal cuff) and direct (strain gauge) blood pressure measurements in 46 anesthetized animals indicated that the metal cuff readings were lower and the cloth cuff readings higher than measurements obtained directly in the same artery with a strain gauge. The blood pressure as measured directly with a strain gauge fell about midway between the corresponding cloth and metal cuff readings.

A Hemorrhagic Syndrome Induced by Derivatives of 3-Hydroxy-1, 4-Naphthoquinone.∗:

Summary 1. A fatal hemorrhagic syndrome accompanied by marked hypoprothrombinemia was produced in rats by the administration of 3 2-substituted-3-hydroxy-1,4-naphthoquinones. 2. These compounds produced only minimal pathologic changes in the liver, caused no measurable destruction of prothrombin in vitro, and possessed only slight bacteriostatic activity against Escherichia coli, the principal source of vitamin K in the gut. 3. The hemorrhagic and toxic properties of at least one of the compounds studied, SN 5090, were antagonized partially by 2 -methyl-1,4-naphthoquinone and completely by vitamin K1. On the basis of this finding it was postulated that these 2-substituted-3-hydroxy-1,4-naphthoquinones, such as SN 5090, compete with the K vitamins in processes essential to prothrombin formation and by displacing these vitamins produce the hemorrhagic syndrome described here.

Dihydrofolate reductase and thymidylate synthetase in strains of Streptococcus faecium resistant to pyrimethamine, chlorguanide triazine, trimethoprim, and amethopterin☆

Abstract Strains of Streptococcus faecium highly resistant to pyrimethamine, chlorguanide triazine or amethopterin exhibited increased levels of dihydrofolate reductase and thymidylate synthetase which varied from 2- to 40-fold. The increases in synthetase were always greater than the corresponding increases in reductase. A strain of the same organism resistant to trimethoprim, however, showed no increases in either enzyme. Studies on the partially purified reductases indicate that the pyrimethamine-resistant enzyme differs strikingly in certain molecular properties. This reductase had a pH optimum at about 5.0 whereas the other four enzymes showed optimum activity at about pH 6.O. It also showed altered inhibition profiles requiring from 4 times as much pyrimethamine to 40 times as much chlorguanide triazine as the sensitive enzyme to obtain 50% inhibition.

Cross-Resistance and Collateral Susceptibility to Antifolic Antimalarial Compounds

Series of strains of Streptococcus faecium ATCC 8043, Lactobacillus casei ATCC 7469, and Pediococcus cerevisiae ATCC 8081 with increasing resistance to the active antifolate antimalarial drugs chlorguanide triazine (CGT), pyrimethamine (PM), and trimethoprim (TMP) were isolated. These mutant strains, stable for at least 3 to 5 years, were examined for cross-resistance and collateral susceptibility to the above compounds and to methotrexate (MTX). Generally, they exhibited cross-resistance to all four compounds, but resistance of a strain to one compound did not predict accurately its resistance to another drug. Unexpectedly, L. casei resistant to CGT exhibited collateral susceptibility to MTX, TMP, and PM varying from 5- to 20-fold. P. cerevisiae developed resistance to CGT readily but maintained its susceptibility to PM and TMP after prolonged exposure to these compounds. Resistance to these antimalarial antifolates was accompanied by only low-grade cross-resistance to MTX, a representative antileukemic antifolate agent.

Antagonism of the hemorrhagic syndrome induced by derivatives of 3-hydroxy-1,4-naphthoquinone.

Summary The development of the hypoprothrombinemia and hemorrhagic lesions, which occurred in the albino rat as the result of ingestion of various 2-substituted-3-hy-droxy-l,4-naphthoquinones, could be prevented either partially or completely by simultaneous administration of 2-methy 1-1,4-naphthoquinone or vitamin Ki. The amounts of these K vitamins required to block development of the above syndrome differed with the various hydroxy-naphthoquinone derivatives. In each instance, however, vitamin Ki proved more effective than 2-methyl-1,4-naphthoquinone. Observations made in this report confirm our earlier suggestion that the hemorrhagic syndrome, which results from administration of 2mdash;substituted — 3-hydroxy-1,4-naphtho-quinones, is due to competition between these substances and the K vitamins in processes involved in prothrombin formation.

Experimental Hypertension in the Rhesus Monkey

Summary Six methods useful in producing experimental hypertension in other laboratory animals were studied in rhesus monkeys. Two methods involving direct constriction of the renal arteries either by silk ligatures or small transparent plastic clamps resulted in a significant incidence of hypertension. Of 13 animals which survived 23 days or longer after renal artery constriction, 10 developed hypertension. The degree of constriction was critical, and a significant proportion of the animals succumbed in acute post-operative renal failure. Four other methods used successfully in rats, rabbits, or dogs were ineffective in the monkey. These included: (1) unilateral temporary renal occlusion and contralateral nephrectomy; (2) unilateral silk perinephritis; (3) unilateral silk perinephritis and contralateral nephrectomy; and (4) bilateral silk perinephritis. Administration of large oral doses of sodium chloride per se did not induce hypertension, nor did it increase the effectiveness of any of the above 4 procedures. Attention has been called to some of the factors which may have contributed to the failure of certain of the methods to produce hypertension in the monkey.

Metabolism of bis(2-chloroethyl)ether and bis(2-chloroisopropyl)ether in the rat

Male rats were given single peroral doses of bis(1-14C-2-chloroethyl)ether ([1-14C]BCEE) (40 mg/kg) and of bis(1-14C-2-chloroisopropyl)ether ([1-14C]BCIE) (90 mg/kg). Excretion of14CO2 and urinary14C was followed for 48 hr. The time required to eliminate one half of the dose was 12 hr for [1-14C]BCEE and 19 hr for [1-14C]BCIE. In the case of [1-14C]BCEE, expired14CO2 accounted for 11.5 ± 5.6(SD) % of the dose, urinary14C accounted for 64.7 ± 14.8%, and 2.4 ± 1.3% was found in the feces. The figures for [1-14C]BCIE were 20.3 ± 9.4% expired as14CO2, 47.5 ± 8.1% as urinary14C, and 3.8 ± 0.3% as fecal14C. Thiodiglycolic acid (TDGA) accounted for roughly 75% of the total urinary14C collected after the [1-14C]BCEE dose. Lesser metabolites of BCEE were 2-chloroethoxyacetic acid (CEAA) (5%), and N-acetyl-S-[2-(2-chloroethoxy)ethyl]-L-cysteine (ACEEC) (7%). Metabolites of [1-14C]BCIE identified in rat urine were 2-(2-chloro-1-methylethoxy)propanoic acid (CMEPA), roughly 36% of the total urinary14C, and N-acetyl-S-(2-hydroxypropyl)-L-cysteine (AHPC) at 19%.