Inhibition of interleukin 2 driven proliferation of mouse CTLL2 cells, by selected carbamate and organophosphate insecticides and congeners of carbaryl.

The anticholinesterase (antiCHE) insecticides, a large family of pesticides used extensively throughout the world, inhibit serine hydrolases by carbamylating or phosphorylating a serine residue at the catalytic site. These insecticides are viewed as potential inhibitors of serine hydrolase-dependent immune functions including interleukin 2 (IL2) signalling. Previous studies in our laboratory have demonstrated that carbaryl (an antiCHE insecticide) produces a marked concentration-dependent inhibition of IL2 driven 1) proliferation of mouse CTLL2 cells, 2) proliferation of human natural killer (NK) cells, and 3) enhancement of target cell killing by human NK cells. In the present study, we examined the potential of 8 antiCHE insecticides (4 carbamates and 4 organophosphates) to inhibit IL2-dependent proliferation of mouse CTLL2 cells. The order of potency for T cell inhibition was carbaryl = dichlorvos > methiocarb > carbofuran > paraoxon > mevinphos > aldicarb = monocrotophos. In view of the relatively high inhibitory potency of carbaryl (a carbamate with low cholinergic toxicity), 3 metabolites and 5 congeners of carbaryl were tested for potency to inhibit CTLL2 proliferation. The data indicate a significant contribution of the 1-naphthol leaving group to inhibition of T cell proliferation by carbaryl, and are consistent with inhibition of a serine hydrolase(s) as a mechanism contributing to the observed inhibition of IL2-dependent proliferation.

Lipid peroxidation in rat liver microsomes: influence of carcinogenic N-nitrosocarbaryl.

The reactivities of carbaryl, N-methyl 1-naphthylcarbamate insecticide and its N-nitrosated derivative carcinogenic, N-nitrosocarbaryl, were investigated on the microsomal hepatic lipid peroxidation and NADPH-dependent reductase activities. The in vivo treatment by N-nitrosocarbaryl produced a reduction in lipoperoxidative degradation induced in vitro by NADPH with regard to the formation of malonaldehyde and conjugated dienes. Carbaryl, its precursor did not affect lipid peroxidation under the same in vivo conditions. Moreover, following administration of the 2 compounds, the activities of NADPH-cytochrome c reductase as well as NADPH-neotetrazolium reductase were significantly decreased by N-nitrosocarbaryl but not influenced by carbaryl. Correspondingly, in vitro studies were performed; different action patterns of the 2 tested xenobiotics were also noted after treatment of rat liver microsomes in vitro by carbaryl and N-nitrosocarbaryl especially in their ability to cope with microsomal oxygen activation. N-Nitrosocarbaryl proved to have a potent inhibitor concentration effect on NADPH-dependent chemiluminescence response in vitro; carbaryl was virtually ineffective on this parameter. No significant difference appeared in the affinity of N-nitrosocarbaryl and carbaryl for the microsomal phospholipids. From the in vitro explorations, it was suggested that carcinogenic N-nitrosocarbaryl may be involved in the inhibition mechanism of microsomal lipid peroxidation through decreases in both NADPH-dependent reductase activities and superoxide generation.

Interaction of carbaryl and N-nitrosocarbaryl with microsomal monooxygenase activities.

The in vitro interactions of carbaryl and carcinogenic N-nitrosocarbaryl with rat liver microsomal monooxygenase activities are compared. The inhibitory effect of the nitroso-compound is demonstrated to be non-competitive on aminopyrine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase. The nature of the inhibition induced by the parent amide is found to be competitive on aminopyrine N-demethylase and p-nitroanisole O-demethylase. Correspondingly, in vitro studies of the metabolism of the two compounds were carried out: they both yield formaldehyde. Moreover, N-nitrosocarbaryl is denitrosated through a NO-cytochrome P-450 complex during microsomal metabolism. The toxic effects and biological activities of the two compounds are discussed on the basis of data of metabolic studies and different patterns of enzyme inhibition.

Local application to mouse skin as a carcinogen specific test system for non-volatile nitroso compounds.

Using the epicutaneous test as an experimental model for detecting carcinogenicity, 3 doses each of nitrosomethylurea (NMU), nitrosonornicotine (NNN) and nitrosocarbaryl (NC) were administered to the skin of 65 female CFLP mice/group. To compare the carcinogenic potency of the nitroso compounds, benzo[a]pyrene (BaP) was taken as reference substance. Dose-response relationships were obtained for NMU and NC as well as for BaP. NNN exhibited only a weak carcinogenic effect in the dose range from 12.5 micrograms to 200 micrograms tested in the skin painting model. It showed, however, no dose dependent activity. After probit analysis of the results, the carcinogenic potencies of the nitroso compounds investigated in this system rank as follows: NC, 0.18; NMU, 0.04; NNN, 0.008 (BaP, 1.00).

Acetylcholinesterase inhibition by sulphur and selenium heterosubstituted isomers of N,N-diethylcarbamyl choline and carbaryl.

Nine sulphur and selenium heterosubstituted isomers of N,N-diethylcarbamylcholine and carbaryl have been prepared and their inhibiting activity towards electric eel acetylcholinesterase (E.C. 3.1.1.7) have been measured. The N,N-diethylcarbamylcholines acted only as reversible inhibitors, i.e. they could not carbamylate the enzyme. The reversible inhibition was of mixed type. Sulphur and selenium substitution had only marginal effects on Ki(0.2-0.3 mM) but reduced the value of K'i. The heterosubstituted carbaryl analogues were, with one exception, found to be irreversible inhibitors, about 100 times less potent than carbaryl.

Comparison of nitrosocarbaryl and carbaryl: in vitro effects on hepatic microsomal aryl hydrocarbon hydroxylase, aminopyrine N-demethylase, epoxide hydrolase and cytosolic glutathione-S-transferase.

The Comparative effects of nitrosocarbaryl and carbaryl on rat hepatic microsomal aryl hydrocarbon hydroxylase, aminopyrine-N-demethylase, epoxide hydrolase and cytosolic glutathione-S-transferase in vitro was investigated. The effects of nitrosocarbaryl on these hepatic drug metabolizing enzyme systems differ markedly (P less than 0.05) from carbaryl. Nitrosocarbaryl was more potent and had much higher inhibitory effect than carbaryl on microsomal aryl hydrocarbon hydroxylase, aminopyrine N-demethylase, epoxide hydrolase and cytosolic glutathione-S-transferase. Inhibition of these enzyme systems by nitrosocarbaryl was dose-dependent. In microsomes, nitrosocarbaryl showed a significant (P less than 0.001) inhibition of aryl hydrocarbon hydroxylase and aminopyrine N-demethylase as compared with the control when the concentration of nitrosocarbaryl added was above 1 X 10(-5) M. The I50 values were 1.8 X 10(-5) M and 3.5 X 10(-5) M, respectively. Nitrosocarbaryl inhibited epoxide hydrolase activity gradually up to 18% at the range of concentration studied (1.3 X 10(-3) M - 1.2 X 10(-2) M) and showed a significant (P less than 0.001) inhibition at the concentration of 1.2 X 10(-2) M. Nitrosocarbaryl at concentrations above 1.6 X 10(-4) M showed a significant (P less than 0.001) inhibition of cytosolic glutathione-S-transferase activity with an I50 value of 3.8 X 10(-4) M. By Contrast, carbaryl failed to inhibit aryl hydrocarbon hydroxylase at concentrations below 3 X 10(-4) M. The pesticide inhibited the enzyme activity only by 20% at the maximum concentration studied (6 X 10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Residues of carbaryl and two of its metabolites in eggs of laying hens treated with Sevin for northern fowl mite control by dipping.

White Leghorn laying hens were treated with carbaryl [Sevin, 1-naphthyl-N-methylcarbamate] by dipping in a .5 or 1.0% (active ingredient) water suspension of an 80% commercially available wettable powder formulation. Residues of carbaryl and two of its metabolites, 1-naphthol and N-hydroxymethyl carbaryl, were detected in eggs within 1 day and reached maximum levels 5 to 7 days after dipping. After that time residues steadily declined but were still detectable 56 days posttreatment in the eggs of the high-dose hens. At no time during the study did total residues in eggs exceed the current .5 ppm egg tolerance for carbaryl and its metabolites.

In vivo formation of nitrosocarbamates in the stomach of rats and guinea pigs.

The N-nitrosocarbamates are potent mutagens and carcinogens, and have been synthesized under acid conditions that prevail in the human stomach. However, it has never been documented that nitrosocarbamates are actually formed in vivo in the stomach of any mammalian species. Using 14C-labeled carbaryl and carbofuran, attempts were made to isolate the nitroso derivatives from the stomach contents of rats and guinea pigs treated orally with the carbamate and sodium nitrite. Only trace quantities of nitrocarbamate were recovered from the rat stomach, whereas 0.5 to 2.0% of the carbamate doses were isolated as the nitroso derivative from the contents of the guinea pig stomach. The rather low apparent yields resulted in part from the instability of the nitrosocarbamates and from absorption of the carbamate and/or nitrosocarbamate from the stomach. Higher rates of synthesis were indicated by incubating the carbamates with sodium nitrite in the presence of the stomach contents at 37 degrees C for 15 min. About 30% nitrosation occurred with the guinea pig and about 0.5% with the rat. The difference was attributed to the pH of the gastric contents. For the rat, the pH ranged from 3 to 5; gastric contents of the guinea pig had a pH between 1 and 2. Since the pH of the human stomach is also in the pH 1-2 range, it is likely that nitrosation of carbamates in humans would be very similar to that in the guinea pig.

Distribution of radioactivity following oral administration of carcinogenic 14CH3-labelled nitrosocarbaryl in the rat.

After a single intragastric administration of 14C-labelled carcinogenic nitrosocarbaryl, a nitrosated pesticide, the distribution of radioactivity was investigated in the blood and a number of organs in male rats. The animals received 0.25 mg/kg of labelled nitrosamine and were killed following administration at timed intervals between 0.5 h and 24 h. Our results show that the greatest amount of the 14CH3--group was associated with the forestomach, tumor-susceptible tissue; the level of radioactivity is noteworthy but less important in the glandular stomach. There are also sites of radioactivity accumulation mainly in the liver. Moreover, [14C]nitrosocarbaryl was revealed in the blood suggesting that nitrosamine itself rapidly (0.5 h) crosses the intestinal barrier and in a significant quantity (13%). These facts constitute a potential carcinogenic risk.

The analysis of N-Nitrosoatrazine and N-Nitrosocarbaryl in whole mice.

N-Nitrosoatrazine and N-Nitrosocarbaryl can be efficiently recovered from the whole mouse by use of a frozen animal procedure in combination with organic solvent extraction. Final qualitative and quantitative analysis is performed by high-performance liquid chromatography in combination with the TEA analyzer (HPLC-TEA). A recovery curve for N-nitrosoatrazine from Swiss CD1 mice indicates an unusually long half-life, especially when compared with more volatile N-nitroso derivatives. These results are of interest in regard to the possible in vivo formation of N-nitroso derivatives of agricultural chemicals.

Inhibition of two rat hepatic microsomal drug-metabolizing enzymes by a carcinogenic N-nitrosated pesticide: N-nitrosocarbaryl.

N-Nitrosocarbaryl (N-methyl-1-naphthyl N-nitrosocarbamate) was intraperitoneally administered to male and female rats on four consecutive days at the following doses: 6.25 mg, 12.5 mg, 25 mg and 50 mg/kg body weight/day in olive oil solution; the controls received just the oil. In a second experiment, a daily intraperitoneal dose of 25 mg/kg of N-nitrosocarbaryl was given for 1, 2, 3 or 4 days; the animals were killed 24 h after the last treatment. The two following microsomal enzymatic activities were assayed: aniline aromatic hydroxylase and p-nitroanisole O-demethylase; the levels of cytochrome P-450, proteins and RNA were measured in the hepatic microsomal fraction. N-Nitrosocarbaryl is an inhibitor of the two investigated microsomal monooxygenases at doses of 25 and 50 mg/kg when administered on 4 consecutive days. During the daily administration, enzyme inhibition is seen in females after one day of treatment whereas cytochrome P-450 only becomes lowered after 4 days of administration. In males, no modification of this parameter is observed whereas the activities of microsomal monooxygenases are inhibited. These results suggest that N-nitrosocarbaryl could act on the active sites of the enzymes which metabolize aniline and p-nitroanisole.

The formation, chemistry and stability of N-nitrosocarbaryl under simulated stomach conditions.

In vitro experiments show it to be very probable that carcinogenic N-nitrosocarbaryl can be formed in the human stomach when carbaryl-contaminated food and nitrites are present. Only in vivo studies can fully elucidate this problem.

Toxicology of various pesticides and their decomposition products on mitochondrial electron transport.

The effect of various pesticides and derivatives on mitochondrial electron transport systems was assessed. DDT, DDE, TDE, Kelthane, chlorobenzilate, chloropropylate and Acarol were found to be inhibitory towards both heavy beef heart mitochondrial (HBHM) NADH-oxidase and succinoxidase enzyme systems. Dichlorobenzophenone and p-chlorophenol were less inhibitory towards the HBHM NADH-oxidase and did not inhibit the succinoxidase enzyme system. DDA did not inhibit either of the electron transport systems. Carbaryl was not inhibitory towards both HBHM oxidase systems, whereas its degradative product dihydroxynaphthalene was inhibitory at the same concentration. Furadan, Matacil, Baygon and Dimetilan were only slightly inhibitory towards themitochondrial NADH-oxidase system and did not inhibit the succinoxidase system. Zectran was inhibitory towards the NADH-oxidase system and was not inhibitory towards the succinoxidase system. DDT, DDE and TDE, dihydroxynaphthalene and 1-naphthol inhibited the NADH-oxidase enzyme system onthe substrate side of cytochrome c, whereas Kelthane inhibited on the oxygen side.

Effect of N-Alkyl chain length on the mutagenicity of N-nitrosated 1-naphthyl N-alkylcarbamates.

1-Naphthyl N-alkylcarbamates (alkyl = methyl, ethyl, n-propyl and n-butyl) were synthesized and then nitrosated with a dichloromethane extract of nitrous acid. The respective N-nitrosated carbamates were purified and tested for mutagenicity using Salmonella typhimurium LT2 strain TA98 and TA100, and Saccharomyces cerevisiae strain D3. The reactivity of the four nitrosocarbamates toward 4-(p-nitrobenzyl)-pyridine (NBP) was measured in order to assess their strengths as alkylating agents. Bioassay results indicated an inverse relationship between the length of the N-alkyl chain and the mutagenicity of the N-nitrosonaphthylcarbamates. The NBP test results indicated an inverse relationship between chemical reactivity and N-alkyl chain length.

Mutagenicity of nitrosocarbaryl and other methylating nitrosamides as related to uptake in Haemophilus influenzae.

Differences greater than 6,000-fold in the mutagenic potency of three nitrosamides that are methylating agents [N-methyl-N-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-nitrosocarbaryl (NC)] could be accounted for in part by differences in their uptake into the Haemophilus influenzae cells. Uptake was roughly correlated with the octanol/water partition coefficient. The most potent mutagen, NC, was concentrated by the cells. The least potent, MNU, was essentially excluded from the bacteria. Uptake was a linear function of concentration, whereas induction of novobiocin-resistant mutations followed higher-order kinetics. The effective dose of chemical to the bacteria (number of molecules taken up) under conditions of mutagenesis could be calculated.

Synthesis and spectroscopic characterization of acetyl-Clx (x = 0,1,2 or 3) and nitroso derivatives of carbaryl and three possible metabolites.

Acetyl-Clx (x = 0,1,2 or 3) and nitroso derivatives of carbaryl, 1-naphthol. 5-hydroxycarbaryl, and 1,5-naphthalenediol were synthesized. Compounds were characterized by infrared, mass, and nuclear magnetic resonance spectroscopy. N-chloroacetylation (Cl = 0,1,2, or 3) of carbaryl was readily accomplished through the pyridine-catalyzed reaction of carbaryl with the appropriate acid chlorides. Trichloroacetylation was also possible through the pyridine-catalyzed reaction with the anhydride. N-nitrosation could also be done readily. N-nitroso- and N-trichloroacetyl carbaryl underwent decomposition to carbaryl on exposure to atmosphere moisture and could be maintained in the pure state only with difficulty. The other derivatives were stable under normal storage conditions.