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With the limited but ongoing usage of perfluorooctane sulfonate (PFOS), the health effects of both PFOS and its alternatives are far from being understood. Long-term potentiation (LTP) was evaluated in rats after exposure to PFOS and its alternatives, aiming to provide some evidence about their potential to affect cognitive ability. Different dosages of PFOS and alternative chemicals, including perfluorohexane sulfonate (PFHxS), perfluorobutane sulfonate (PFBS) and chlorinated polyfluorinated ether sulfonate (Cl-PFAES), were given to rats viaacute intracerebroventricular injection. The field excitatory postsynaptic potential (fEPSP) amplitude of the input/output functions, paired-pulse facilitations, and LTP in vivowere recorded. PFOS and its alternatives inhibited LTP in varying degrees, without significant effects on the normal synaptic transmission. In addition, PFHxS and Cl-PFAES exhibited comparable potential to PFOS in disturbing LTP. The results suggested that acute exposure to PFOS and its alternatives impaired the synaptic plasticity by a postsynaptic rather than a presynaptic mechanism. Besides, the fEPSP amplitude of the baseline was reduced by Cl-PFAES but not by other compounds, indicating that Cl-PFAES might act in a different mode. Providing some electrophysiological evidence and the potential mechanism of the neurotoxicity induced by PFOS and its alternatives, the present study addresses further evaluation of their safety and health risks.

We studied the embryonic and maternal genotoxicity of pyrimethamine (PYR), a potent teratogen and folate antagonist, using alkaline single cell gel electrophoresis (SCG, or Comet) assay as modified by us (we used isolated nuclei instead of isolated cells). ICR mice were treated on the 13th day of pregnancy with a single oral dose of 50 mg PYR/kg. Six maternal organs (liver, kidney, lung, brain, spleen, bone marrow), maternal and fetal placentas, and two embryos were taken 6 and 16 h after treatment; the embryos were divided into head and body portions. Each sample was minced, homogenized gently, and centrifuged. The nuclei from the precipitates were used. PYR induced DNA damage in all maternal organs except spleen and bone marrow 6 h after administration. The DNA damage in all the affected organs was less at 16 h than at 6 h, and that of the kidney and brain returned to control level at 16 h. PYR also induced DNA damage in maternal and fetal placentas and embryos that was detected at 6 and 16 h, with greater damage at 6 h. Co-treatment of folinic acid calcium salt (FNA, 10 mg/kg ip), a reduced active folate form, prevented the PYR-induced DNA damage in all target tissues examined 6 h after treatment. These data indicate that the observed embryonic and maternal DNA damage caused by PYR may be related to folate deficiency, and that the modified alkaline SCG assay can be used to predict fetal/embryonic genotoxicity in vivo, in addition to the organ-specific maternal genotoxicity.

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of four hydrazine derivatives—1,2-dimethylhydrazine (SDMH), 1,1-dimethylhydrazine (UDMH), hydrazine (HZ), and procarbazine (PCZ)—in mouse liver, lung, kidney, brain, and bone marrow, and in the mucosa of stomach, colon, and bladder. Mice were sacrificed 3 and 24 h after intraperitoneal (i.p.) and oral (p.o.) administration. SDMH at 20 mg/kg i.p. yielded statistically significant DNA damage in all tested organs except for lung. In the gastrointestinal tract, SDMH was genotoxic in the stomach and the colon after i.p. treatment but only in the colon after 20 and 30 mg/kg p.o. treatment. UDMH at 50 mg/kg i.p. yielded DNA damage in the liver and lung at 3 h. PCZ at 200 mg/kg i.p. caused DNA damage in the liver, kidney, lung, brain, and bone marrow. UDMH and PCZ were positive in the stomach and colon p.o. but not by i.p. treatment. HZ at 100 mg/kg yielded DNA damage in the stomach, liver, and lung when given i.p. and in the brain when p.o. Thus, the administration route is important when evaluating organ-specific genotoxicity in multiple organs.

Potentiation of Acute Toxicity of 2-sec-Butylphenyl N-methylcarbamate (BPMC) by Fenthion in Mice. MIYAOKA, T., TAKAHASHI, H., TSUDA, S., AND SHIRASU, Y. (1984). Fundam. Appl. Toxicol. 4, 802–807. This study was undertaken to determine whether interactions of toxicologic importance might occur during combined exposure of male mice to 2-sec-butylphenyl N-methylcarbamate (BPMC) and O,O-dimethyl O-(3-methyl-4-methylthiophenyl)phosphorothioate (fenthion). The equitoxic (administration of BPMC and fenthion resulted in only a 1.6-fold potentiation compared to the expected LD50. However, 1 hr oral pretreatment with 3 (1/100 LD50), 7.5, 15, and 30 mg/kg of fenthion resulted in 5-, 9-, 12-, and 15-fold potentiation of the acute oral toxicity of BPMC, respectively. This fenthion pretreatment caused significant increases in the BPMC plasma concentrations and in the area under the concentration-time curve. The increase of the plasma concentrations of BPMC depended upon the fenthion dose and was associated with that of the potency ratio. The pretreatment of fenthion prolonged the hexobarbital sleeping time. The plasma concentrations of BPMC after the administration of a dose of LD2.5 (20 mg/kg) in fenthion-pretreated mice were clearly lower than those of a dose of LD2.5 (195 mg/kg) in nontreated mice. These results suggested that fenthion pretreatment caused the potentiation of the acute toxicity of BPMC partially by the inhibition of detoxification of BPMC.

Potentiated Toxicity of 2-sec-Butytpbenyl Methylcarbamate (BPMC) by O,O-Dimethyl O-(3-Methyl-4-nitrophenyl)phosphorothioate (Fenitrothion) in Mice; Relationship between Acute Toxicity and Metabolism of BPMC. TAKAHASHI, H., MIYAOKA, T., TSUDA, S., AND SHIRASU, Y. (1984). Fundam. Appl. Toxicol. 4, 718–723. Fenitrothion of oral subtoxic dose (100 mg/kg; 4 hr pretreatment) decreased acute oral LD50 of BPMC from 360 to 66 mg/kg in male mice. The treatment prolonged the hexobarbital sleeping time and increased the plasma BPMC concentrations. The BPMC toxicity and its plasma concentrations were significantly reduced by phenobarbital treatment (80 mg/kg/day, 2 days, ip). This treatment diminished the effects of fenitrothion on BPMC toxicity and plasma BPMC concentrations. BPMC was metabolized by mixed-function oxidases of the liver in vitro. The metabolism of BPMC was competitively inhibited by the addition of fenitrothion (5 μg/ml). Fenitrothion remained in the liver (7 μg/g liver). These results suggest that competitive inhibition of BPMC metabolism by fenitrothion may, at least in part, play a role in inhibition of BPMC detoxication, resulting in potentiation of its toxicity.

Pharmacokinetk Analysis of Increased Toxicity of 2-sec-Butylphenyl Methylcarbamate (BPMC) by Fenitrothion Pretreatment in Mice. TSUDA, S., MIYAOKA, T., IWASAKI, M., AND SHIRASU, Y. (1984). Fundam. Appl. Toxicol. 4, 724–730. The potentiating effect of O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate (fenitrothion) on the toxicity of 2-sec-butylphenyl methylcarbamate (BPMC) in male mice was analyzed pharmacokinetically. The animals pretreated by dietary administration of 1000 ppm fenitrothion for 1 week (4.4% of the po LD50 daily) did not show toxic symptoms except for a slight decrease in body weight In the fenitrothion-pretreated mice, toxicity of fenitrothion was not changed but a fivefold potentiation was observed in po and ip acute lethality and a threefold potentiation of iv lethality of BPMC. Toxic signs after BPMC administration were similar regardless of fenitrothion pretreatment or of route of administration. Fenitrothion pretreatment followed by BPMC administration (20 mg/kg po or 8 mg/kg iv, approximate LD5 in the pretreated mice) significantly increased the plasma BPMC concentration and the total area under the plasma concentration versus time curve (AUG0-∞. The pretreatment increased the oral AUC0-∞, more greatly than the iv AUC0-∞, (for po, 6.3-fold; for iv, 2.0-fold). The oral systemic availability of BPMC (fraction reaching systemic circulation) was increased by fenitrothion treatment to 3.3-fold. These results suggest that a major cause of the potentiation may be the increase in amount of BPMC in the systemic circulation.

Potentiations of N-Methylcarbamate Toxicities by Organophosphorus Insecticides in Male Mice. TAKAHASHI, H., KATO, A., YAMASHITA, E., NAITO, Y., TSUDA, S., and SHIRASU, Y. (1987). Fundam. Appl. Toxicol. 8, 139–146. A N-methylcarbamate insecticide, 2-sec-butylphe-nyl N-methylcarbamate (BPMC), is markedly potentiated by low-dose treatments of P=S type organophosphorus insecticides. As a mechanism of this potentiation, the increase of plasma BPMC concentrations due to the inhibited metabolic degradation has been suggested. In this study, acute toxicities of five N-methylcarbamates structurally related to BPMC were studied after low-dose treatments of three P=S type organophosphorus insecticides (cyanophos, feni-trothion, and malathion) and one P=O type organophosphorus insecticide (dichlorvos), and the role of plasma concentrations of N-methylcarbamates in the potentiations was examined. Acute toxicities of five N-methylcarbamates were potentiated by the treatments of the P=S types, among which the potentiation of BPMC was strongest. BPMC toxicity was not potentiated by the treatment of the P=O type. Plasma concentrations of BPMC were increased by the treatments of the P=S types, but not by the treatment of the P=O type. The acute toxicity and plasma concentrations of BPMC were increased by SKF 525-A (an inhibitor of mixed-function oxidase). These results suggest that the increase of plasma BPMC concentrations may be related to the potentiation of BPMC toxicity. The treatment of fenitrothion increased plasma concentrations of other N-methylcarbamates more than those of BPMC, although the potentiation of BPMC toxicity was strongest. SKF 525-A and fenitrothion treatments increased plasma BPMC concentrations to a similar degree, but the potentiation of BPMC toxicity by SKF 525-A was significantly less than that by fenitrothion. Thus, some other mechanism(s) may be responsible for the potentiations of the N-methylcarbamate toxicities.

Contribution of Monoaminergic Nervous System in Potentiation of 2-sec-Butylphenyl N-Methylcarbamate (BPMC) Toxicity by Malathion in Male Mice. TAKAHASHI, H., TANAKA, J., TSUDA, S., and SHIRASU, Y. (1987). Fundam. Appl. Toxicol. 8, 415–422. Malathion-induced marked potentiation of BPMC toxicity (about fivefold) was analyzed by measuring LD50 as an index of acute toxicity. The acute lethality of BPMC was decreased by muscarinic blockers (atropine, methylatropine, or trihexyphenidyl) or a monoamine oxidase inhibitor (pargyline) and increased by a monoamine depleter (reserpine) or a dopaminergic blocker (haloperidol). The potentiation observed with BPMC and malathion was decreased by the muscarinic blockers, monoamine depleters (reserpine, α-methyl-p-tyrosine), an α-noradrenergic blocker (phen-tolamine), or haloperidol. The acute toxicities of other N-methylcarbamates MPMC (3,4-di-methylphenyl N-methylcarbamate), MTMC (3-methylphenyl N-methylcarbamate), NAC (1-naphthyl N-methylcarbamate), and XMC (3,5-dimethylphenyl N-methylcarbamate) were potentiated by malathion to a lesser degree than that of BPMC. Atropine protected against the lethalities of all N-methylcarbamates. Reserpine or haloperidol potentiated the lethalities of N-methylcarbamates with a similar tendency toward malathion. When the inhibitory effect of each N-methylcarbamate on brain acetylcholinesterase (AChE) was compared with its LD50, among five N-methylcarbamates BPMC had particularly strong anti-AChE activity. This characteristic of BPMC was not observed after the treatment with reserpine. These results suggest that BPMC may act not only on cholinergic nerves as an anti-AChE, but also on monoaminergic nerves which antagonize the lethal cholinergic effect. Malathion might inhibit the effect of BPMC on the monoaminergic nerves, thereby markedly potentiating the lethal effect of BPMC.

Metabolic Induction of the Hepatic Cytochrome P450 System by Chlorfenvinphos in Rats. IKEDA, T., TSUDA, S., AND SHIRASU, Y. (1991). Fundam. Appl. Toxicol. 17, 361–367. Previous studies have shown that a single oral pretreatment of rats with the organophosphorus insecticide 2-chloro-l-(2,4-dichlorophenyl)vinyl diethyl phosphtate (chlorfenvinphos, CVP) afforded protection against the toxicity of a subsequent challenge with the same compound within 24 hr. This protection may be due to the reduction in brain cholinesterase inhibition caused by the decrease in plasma CVP concentration. The purpose of this study was to investigate the mechanism of the decrease in plasma CVP concentration in relation to metabolic induction. CVP was preferentially metabolized by a liver microsomal fraction with an NADPH-generating system, compared with serum or kidney subcellular fractions. A single oral 24-hr pretreatment with CVP (15 mg/kg) increased the oral LD50 of its next dosage to threefold. The same treatment also increased CVP metabolism (to 178%), cytochrome P450 content (to 130%), cytochrome P450 reductase activity (to 130%). cytochrome b5 content (to 121%). and cytochrome P450-linked activities such as aminopyrine demethylase (to 140%) and aniline hydroxylasc (to 127%) in the hepatic microsoma1 fraction. A single oral 24-hr pretreatment of phenobarbital (50 mg/kg), which is known as an inducer of cytochrome P450, increased the oral LD50, of CVP and all the related metabolic parameten listed above in an order of magnitude similar to that of CVP, although the increments induced by the phenobarbital treatment were greater than those induced by the CVP treatment These results indicate that the increase in hepatic CVP metabolism may be due to the induction of the hepatic cytochrome P450 system caused by the single oral short-term treatment with CVP. This induction may be one of the reasons for the deaertse in plasma CVP concentration which may be responsible for the reduction in toxicity of its next dosage.

Pretreatment of Rats with an Organophosphorus Insecticide, Chlorfenvinphos, Protects against Subsequent Challenge with the Same Compound. IKEDA, T., KOJIMA, T., YOSHIDA, M., TAKAHASHI, H., TSUDA, S., AND SHIRASU, Y. (1990). Fundam. Appl. Toxicol. 14, 560–567. A single oral pretreatment of rats with chlorfenvinphos (CVP) reduced toxicity of the same compound subsequently administered. This protection occurred 8 hr and became maximal 24 hr after the oral pretreatment at a dose of 15 mg/kg (about half of its LD50). The 24-hr pretreatment with CVP increased the LD50 of CVP threefold, but did not change the type of toxic signs and time to death caused by CVP. The CVP pretreatment did not appreciably change the toxicities of the cholinergic agonists, carbachol and oxotremorine, but significantly increased the toxicity of another organophosphate, dichlorvos. Oral treatment of rats with CVP (15 mg/ kg) inhibited brain acetylcholinesterase (AChE) activity. This inhibition became maximal at 4 hr (about 20% of control) and lasted more than 24 hr after the administration. Twenty-four hours after oral administration of CVP (15 mg/kg), the second dose (CVP 30 mg/kg, po) was less effective in inhibiting cholinesterase activities of the brain, erythrocyte, and plasma compared with naive rats treated with the same dose. The difference in brain AChE activity between control and CVP pretreatment groups was greater in magnitude than that measured in erythro-cytes. CVP concentration in plasma after the oral administration of CVP (30 mg/kg) was decreased by the CVP pretreatment. Area under the concentration vs time curve (AUC) in the CVP-pretreated group was about one-fourth of AUC in the control group. This decrease in the AUC was comparable to the decrease in the toxicity of CVP. Thus, the protection against subsequent CVP challenge may be due to the reduction in the inhibition of brain AChE activity caused by the decrease in plasma CVP concentration.

We have previously reported that the acute oral toxicity of chlorfenvinphos (CVP) is reduced by the oral pretreatment of rats with the same compound. In this report, the mechanism of this protection was clarified mainly through the physiologically based pharmacokinetic analysis. The CVP pretreatment (15 mg/kg, po, 24 hr before) reduced the lethality of po CVP greatly, and that of iv CVP to a lesser extent. Brain acetylcholinesterase inhibition by po and iv CVP was also decreased by the pretreatment. The magnitude of reduction of the inhibition caused by the po CVP was greater than that of the iv CVP. The ratio CVP concentration between the brain and plasma was the same, regardless of the route of administration or the pretreatment. The pretreatment greatly reduced the plasma concentration and the area under the plasma concentration versus time curve (AUC) of the po CVP, but did not change appreciably that of the iv CVP. The unbound fraction of CVP in the blood or the liver was not changed by the pretreatment. According to physiologically based pharmacokinetic analysis, the decrease in AUC of the po CVP may be mainly caused by an increase in intrinsic clearance of the liver and a decrease in the partition coefficient of CVP between the emergent blood and the liver. The increase in the intrinsic clearance may be related to the metabolic induction observed in vitro. The pretreatment decreased the absorption rate constant of the po CVP. This change in combination with the above two factors which reduce AUC might be the reason for the decrease in the plasma concentration after the po CVP, and the protection against the CVP toxicity of the succeeding dosage.

Differences in the Mode of Lethality Produced through Intravenous and Oral Administration of Organophosphorus Insecticides in Rats. TAKAHASHI, H., KOJIMA, T., IKEDA, T., TSUDA, S. and SHIRASU, Y. (1991). Fundam. Appl. Toxicol. 16, 459–468. This study was undertaken to investigate the possibility that mechanisms other than cholinesterase (ChE) inhibition account for the acute toxicity of organophosphorus insecticide. Both the P═O type insecticide (direct ChE inhibitors: chlorfenvinphos and dichlorvos) and the P═S type insecticide (indirect ChE inhibitors: diazinon and fenthion) were employed. Rats treated with lethal doses of intravenous and oral P═O type insecticides and oral P═S type insecticides exhibited typical signs of anti-ChE poisoning along with marked inhibition of brain and erythrocyte ChE activity. In contrast, rats given lethal doses of intravenous P═S type insecticides exhibited tonic convulsions and opisthotonos, with only slight inhibition of ChE activities. When P═O type insecticides were intravenously administered to anesthetized and conscious rats, animals exhibited typical anti-ChE poisoning signs in cardiorespiration: hypertension and apnea which were antagonized by atropine. After administration of lethal doses of P═O type insecticides, breathing disappeared before the cessation of heart beats. Rats receiving lethal doses of intravenous P═S type insecticides did not show hypertension, but exhibited transient cessation of breathing and heart beats. Breathing was observed after the disappearance of heart beats. The electroencephalogram (EEG) was characterized by spike and wave complexes. The EEG and cardiorespiratory changes were not antagonized by atropine. It was concluded that lethality following intravenous P═S type insecticides may be independent of ChE inhibition.

We studied the combined toxicity of organophosphates (Cyanophos, Dichlorvos, Fenitrothion and Malathion) and carbamates (BPMC, MPMC, MTMC, NAC and XMC) in male mice. In the study of 8 equitoxic mixtures, a marked potentiation was noted with malathion and BPMC (5.4-fold). The toxicity of BPMC was markedly potentiated by subtoxic treatment with organophosphate, Cyanophos, Fenitrothion or Malathion, which were metabolized through oxidative desulfuration, but not by Dichlorvos which avoids the reaction. The 3 treatments which enhanced BPMC toxicity increased the toxicity of other carbamates with less potency. The potentiation ratios of the 5 carbamates after each of the 3 treatments were significantly correlated to one another, suggesting common mechanisms responsible for the potentiation. Thus, it could be explained in part by the inhibitory effect of organophosphate through oxidative desulfuration on carbamate detoxication. The potentiation of Malathion and BPMC was decreased to 1.9-, 1.9-, 3.4-, and 2.4-fold by atropine, trihexyphenidyl, methylatropine, and reserpine treatment, respectively. Consequently, cholinergic (especially central) and monoamine neurons were also suggested to be involved in part in the potentiation. The interaction in AChE level was excluded for the potentiation.

Inhalation Chamber with Size Discriminator for Liquid Aerosols. TSUDA, S., IWASAKI, M., YOSHIDA, M., AND SHIRASU, Y. (1984). Fundam. Appl. Toxicol. 4, 378–387. To minimize data variation in inhalation toxicity testing and to evaluate human and animal hazards of inhaled chemicals, a practical inhalation chamber with a size discriminator for mists was developed to provide high concentration liquid aerosols of denned particle sizes. Liquid aerosols generated with an atomizer were separated by an impinging separator which was composed of aerosol jets directed upward against a flat plate. The principle of the separator eliminates particles larger than a calculated cutoff size in micrometer and submicrometer ranges by changing the orifice diameter of the jet nozzle under constant air flow. The mists thus separated are introduced into the space between two concentric cylinders just above the impaction plate. Ten rats can be positioned around the periphery of the chamber all equidistant from the impaction plate, with their snouts thrust into the inhalation space. Preliminary testing with olive oil and water aerosols using particle cutoff sizes of 1, 3, and 3.3 μm showed that the obtained separation of particles was very clear, although the cutoff point seemed to shift somewhat to smaller values than calculated; the shift was especially evident with water aerosols. The concentrations obtained were more than 1 mg/liter when the cutoff point was selected at 1 μm. The mist at the inhalation space attained a steady concentration and particle size distribution within 2 min of the onset of mist injection, remained over a 4-hr period, and was cleared within 2 min of the cessation of mist generation.