Cytokine induction by a linear 1,3-glucan, curdlan-oligo, in mouse leukocytes in vitro.

OBJECTIVE: Curdlan, an extracellular bacterial polysaccharide, is a linear beta-1,3-glucan. Previously, we developed Curdlan-oligo (CRDO). We investigated its effect on the production of cytokines in leukocytes from mice, and compared its activity with that of SCG, a 6-branched 1,3-beta-glucan. METHODS: Splenocytes from DBA/2 mice were cultured with CRDO or SCG (0, 1, 10 or 100 microg/ml) in vitro, and then the supernatants were collected to measure cytokines. Bone marrow-derived dendritic cells (BMDCs) were cultured with CRDO (0, 1, 10 or 100 ng/ml) in vitro, and then the supernatant was collected to measure cytokines. RESULTS: SCG stimulated splenocytes in DBA/2 mice to produce GM-CSF, IFN-gamma and TNF-alpha. CRDO induced production of GM-CSF and IFN-gamma, but not TNF-alpha. The amounts of GM-CSF and IFN-gamma were small compared with those produced in response to SCG. The effect of SCG on TNF-alpha production was partially inhibited by CRDO. In bone marrow-derived dendritic cells, CRDO induced production of TNF-alpha and IL-6. CONCLUSION: Taken together, these results suggest that CRDO stimulated mouse leukocytes to induce the production of cytokines, and the mechanism of the effect of CRDO on leukocytes is different from that of SCG.

Molecularly imprinted chiral stationary phase prepared with racemic template.

The first example of molecularly imprinted chiral stationary phase prepared using a racemic template is shown. N-(3,5-Dinitrobenzoyl)-α-methylbenzylamine (DNB) was chirally discriminated on the molecularly imprinted stationary phase prepared using racemic DNB as the template. A chiral monomer, (S)-(-)-N-methacryloyl-1-naphthylethylamine, was utilized as the functional monomer toward the racemic template, and its chiral recognition ability was, interestingly, found to be enhanced through racemic molecular imprinting. A thermodynamic discussion briefly suggests that the observed chiral recognition ability of the racemic imprinting was proper value.

Bio-anticlastogenic effects of unsaturated fatty acids included in fish oil--docosahexaenoic acid, docosapentaenoic acid, and eicosapentaenoic acid--in cultured Chinese hamster cells.

Bio-anticlastogenic effects of unsaturated fatty acids--cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), cis-7,10,13,16,19-docosapentaenoic acid (DPA), and cis-5,8,11,14,17-eicosapentaenoic acid (EPA)--on chemically induced chromosome aberrations were studied in cultured Chinese hamster cells. The induction of chromosome aberrations by the crosslinking agents mitomycin C (MMC) and cisplatin (DDP), the SN-1 type alkylating agents N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl nitrosourea (MNU), and ethyl nitrosourea (ENU), and the SN-2 type alkylating agent ethyl methanesulfonate (EMS), but not by the SN-1 type alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the SN-2 type alkylating agent methyl methanesulfonate (MMS), was suppressed by post-treatment with DHA, DPA, and EPA. Since there was no opportunity to inactivate mutagens by desmutagenic mechanisms under the post-treatment schedule used, the results demonstrate the bio-anticlastogenicity of unsaturated fatty acids. Suppression by the unsaturated fatty acids was observed when cells were treated during the G2 phase, suggesting that G2 events were responsible for the bio-anticlastogenic effects. Two saturated fatty acids with the same number of carbons as the studied unsaturated fatty acids--docosanoic acid and eicosanoic acid--did not affect chromosome aberration induction, suggesting the necessity of unsaturation for fatty acid bio-anticlastogenicity.

Suppressing effects of S phase post-treatment with carbolines on sister-chromatid exchanges induced by mitomycin C in Chinese hamster cells.

The effects of post-treatment with four types of carbolines-alpha-, beta-, gamma-, and delta-carbolines-and aminoimidazoazaarenes during S phase on the frequency of SCEs induced by MMC were studied in synchronized Chinese hamster CHO cells. Post-treatment with alpha-carbolines (A alpha C, MeA alpha C), beta-carbolines (harman, norharman, harmine, and harmaline), gamma-carbolines (Trp-P-1 and Trp-P-2) and delta-carbolines (Glu-P-1, and Glu-P-2) during S phase at non-clastogenic concentrations caused a statistically significant decrease in the frequency of SCEs induced by MMC. Aminoimidazoazaarenes (IQ, MeIQ, and diMeIQx) showed no effects on SCEs, suggesting that the SCE-suppressing activity resides in the carboline structure. The data suggest that the suppressing effect may be due to inhibition of DNA replication.

Inhibitory effect of fenthion and diazinon on the contraction of rat aorta, and its contribution to lethality.

Fenthion and diazinon, P = S type organothiophosphates which are precursors of cholinesterase inhibitors, cause remarkable atropine-insensitive hypotension in rats when administered intravenously in lethal doses. We investigated their effects on isolated rat aorta and atria to reveal the site of action. Fenthion and diazinon inhibited both types of contractions induced by high K+ solution and norepinephrine in aortic preparations from which the endothelium was removed. IC50 values (under [Ca2+] = 1.5 mM) were 2 x 10(-5) M and 7 x 10(-5) M, respectively. However, the atrial preparations were relatively resistant, since fenthion showed no effect up to 10(-3) M and diazinon at 10(-4) M exhibited a slight inhibition which was antagonized by atropine. The hypotensive effect of fenthion or diazinon was therefore attributable to the direct inhibiting action on the arterial muscle tone, which may be independent of the activation of muscarinic receptors. The results suggested that fenthion and diazinon affect movement and/or utilization of calcium in the aortic muscle cells, since an increase in the calcium concentration in the bathing solution antagonized their inhibitory effect.

Enhancing effects of heterocyclic amines and beta-carbolines on the induction of chromosome aberrations in cultured mammalian cells.

The effects of post-treatment with heterocyclic amines and beta-carbolines on the induction of chromosome aberrations were studied in Chinese hamster CHO K-1 cells and SV40-transformed excision repair-deficient human XP2OSSV cells. The number of chromosome aberrations induced by UV and MMC were increased by post-treatment with Trp-P-1 and Trp-P-2, in both the presence and the absence of S9 mix. A alpha C, MeA alpha C, Glu-P-1, Glu-P-2, IQ, MeIQ, harman and harmine increased chromosome aberrations only in the presence of S9 mix. Glu-P-2, IQ, MeIQ, harman, and harmine did not induce chromosome aberrations by themselves at the concentrations used in this study. Trp-P-1, Trp-P-2, A alpha C, MeA alpha C and Glu-P-1 were weak clastogens by themselves, but at much higher concentrations than those at which they increased the induction of chromosome aberrations in cells pretreated with UV or MMC. Therefore, the increases in chromosome aberrations were not considered to be additive.

Non-cholinergic mechanisms underlying the acute lethal effects of P = S type organophosphorus insecticides in rats.

Intravenous administration of the lethal dose of diazinon or fenthion, P = S type organophosphates, to urethan anesthetized rats induced bradycardia and transient apnea followed by a decline of blood pressure, and death. We investigated the mechanisms of the lethal action of these organophosphates in rats through measurements of blood pressure, heart rate, and respiratory pattern. We compared their cardiorespiratory effects in the five different conditions under anesthesia; 1) normal (without treatment), 2) artificially ventilated, 3) vagotomized, 4) atropinized, 5) pithed, vagotomized and atropinized. It was found that the administration of 200 mg/kg of fenthion and 100 mg/kg of diazinon, caused sudden bradycardia, transient apnea and gradual decline of blood pressure in the anesthetized normal rat, and the rat died. The rats in other conditions also died except the artificially ventilated rats, in which 400 mg/kg of fenthion was administered to cause hypotension and subsequent death. Hypotension was observed consistently even after the cardiac effect such as bradycardia was eliminated by atropine treatment. In the pithed rats which were further vagotomized and atropinized, these organophosphates also caused hypotension. These results may indicate that hypotension is the main cause of death which resulted from intravenous administration of the P = S types. Hypotension may be caused by peripheral cardiovascular effect of the P = S types, which is unrelated to cholinergic mechanisms.

Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C.

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclass of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the clastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for the detection of the clastogenic potential of promutagenic chemicals.

Pharmacokinetic analysis of protection by an organophosphorus insecticide, chlorfenvinphos, against the toxicity of its succeeding dosage in rats.

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 of 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.

Polydactyly lethal: a new mutant spontaneously occurring in the FPL strain of rats.

A new mutant gene that causes preaxial polydactyly in the hindlimbs was found in the strain of rats with fused pulmonary lobes (fpl). Genetic analysis has revealed that the new mutation is inherited as an autosomal recessive trait and is not closely linked with the fpl gene. Since homozygous mutants die within the first 2 days after birth, the mutant gene was named polydactyly lethal, gene symbol pl. A test for allelism between the pl gene and another gene, pd, which also causes preaxial duplication anomalies, showed no allelism between these two genes. Skeletal examination revealed that all pl/pl newborns had thickening and/or bifurcation of tarsal I and metatarsal I, as well as duplication of the proximal and distal phalanges of digit I in the hindlimbs. In some cases, phalangeal duplication or bifurcation in digit I with thickening of metacarpal I was also found in the forelimbs, although extra forelimb digits were not detected externally. The pl/pl newborns showed hunchback-like abnormal posture externally and had several associated vertebral abnormalities in varying degrees, i.e., kyphosis, scoliosis, splitting of the thoracic vertebral bodies, and fusion of the lumbar vertebral bodies. No major malformations were seen in the visceral organs. The cause of neonatal deaths has not yet been determined.

Metabolic induction of the hepatic cytochrome P450 system by chlorfenvinphos in rats.

Previous studies have shown that a single oral pretreatment of rats with the organophosphorus insecticide 2-chloro-1-(2,4-dichlorophenyl)vinyl diethyl phosphate (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 hydroxylase (to 127%) in the hepatic microsomal 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 parameters 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 decrease in plasma CVP concentration which may be responsible for the reduction in toxicity of its next dosage.

Effects of the Ay gene on susceptibility to hydrocortisone fetotoxicity and teratogenicity in mice.

Effects of the Ay gene, a coat color gene, on susceptibility to hydrocortisone fetotoxicity and teratogenicity were investigated by using the congenic strain of C57BL/6-Ay (Ay/a) which had been maintained by repeated back-crosses of the Ay gene to the C57BL/6 (a/a) background. Matings were conducted as follows (female x male): group I, a/a; group II, a/a x Ay/a; and group III, Ay/a x a/a. Pregnant females were subcutaneously given daily doses of 0, 12.5, 25, or 50 mg/kg of hydrocortisone on days 10-13 of pregnancy. On day 18 of pregnancy, fetuses were sexed, weighed, and examined for external abnormalities. In group I, the mean fetal weight was significantly decreased at a dose of 25 mg/kg or more. The incidences of cleft palate were 3.2 and 22.7% at 25 and 50 mg/kg, respectively. In group II, in which half of the fetuses were expected to carry the Ay gene, the mean fetal weight was decreased significantly at 12.5 mg/kg or more. The incidence of cleft palate in group II at 50 mg/kg was 44.2%, which was significantly higher than that in group I. In group III, in which maternal mice as well as half of their fetuses carried the Ay gene, a decrease in the mean fetal weight was greater than in group II. In addition, the mean percentage of fetal resorptions was significantly increased at 50 mg/kg. The incidence of cleft palate in group III was significantly increased at 25 mg/kg (10.5%) when compared with those in groups I and II. These results indicate that the Ay gene may be associated with susceptibility to hydrocortisone fetotoxicity and teratogenicity in mice.

Chronic toxicity studies with thiram in Wistar rats and beagle dogs.

Groups of 64 male and 64 female Wistar rats were given thiram at constant dietary doses of 0, 3, 30, and 300 ppm (0, 0.1, 1.2, and 11.6 mg/kg/day for males and 0, 0.1, 1.4, and 13.8 mg/kg/day for females) for 104 weeks. Eight males and eight females in each group were killed after Weeks 13, 26, and 52. For the dog study, four male and four female beagle dogs were alloted to each group and treated with the compound at 0, 0.4, 4, and 40 mg/kg/day for 104 weeks. The dogs in the 40 mg/kg/day group had severe toxic signs, including nausea or vomiting, salivation, and occasional clonic convulsion, and all were subjected to unscheduled necropsy before Day 203 of treatment. The dogs also had ophthalmological changes such as fundal hemorrhage, miosis, and desquamation of the retina which were consistent with the retinal lesions shown by histopathology. The rats of the high-dose group had retarded growth with a slightly decreased food intake. Anemia was evident in high-dose female rats and in middle- and high-dose dogs. Liver failure in male and female dogs and kidney damage in female dogs were detected in middle- and high-dose groups by blood biochemistry and/or histopathology. Regressive changes of the sciatic nerve accompanied by atrophy of the calf muscle were seen in female rats of the high-dose group but not in male rats. In high-dose rats, progression of myocardial lesions of the heart and chronic nephrosis of the kidney were depressed in males and females, respectively. Female rats of the middle- and high-dose groups had decreased occurrences of mammary fibroadenoma and decreased development of skin masses.

Amelanotic melanocytic tumors of the pinna in six F344 rats.

Spontaneous amelanotic melanocytic tumors of the pinna were found in six females of 960 male and 960 female albino (F344/DuCrj) rats which had been used in three different 24-month chronic toxicity studies. The age when the pinnal tumors were detected ranged from 37 to 59 weeks. The tumors were located unilaterally in the pinna and observed as subcutaneous spherical to irregular, solid white masses measuring 7 to 25 mm in diameter. The pinnal tumors were histologically classified into spindle cell and pleomorphic cell types. The spindle cell type was observed in four rats and composed of fusiform cells arranged in interlacing bundles. The pleomorphic cell type was observed in the remaining two rats and composed of pleomorphic large cells arranged in sheets. One tumor of the latter type metastasized to the submaxillary lymph node and lung. Melanin pigments were not demonstrated in any of the tumors. In immunohistochemistry, nuclei and cytoplasm of tumor cells in all the tumors were slightly positive for S-100 protein. Ultrastructurally, tumor cells contained a considerable number of premelanosomes in the cytoplasm. Desmosomes were occasionally observed between the cell membranes of the adjacent tumor cells. No distinct basal lamina was seen around tumor cells.

Differences in the mode of lethality produced through intravenous and oral administration of organophosphorus insecticides in rats.

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.

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice.

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.

Male infertility in the hereditary polydactyly (PD) rat.

In the PD strain of rats, male pd homozygotes never sire, whereas male heterozygotes and female hetero- and homozygotes are fertile. In order to investigate the cause of infertility, pd/pd males were examined for their reproductive ability and gross and histological changes in the reproductive organs. Mating ability of pd/pd males was comparable to that of pd/+ males when a 2 week cohabitation procedure was employed. Cryptorchidism, which is considered to be a cause of infertility, was observed in 63% of the 12 week old pd/pd males. However, this abnormality frequently was observed unilaterally and the incidence of bilateral cryptorchidism was only 13%. Although the relative weights of descended testes and epididymides were comparable between the pd/pd and pd/+ males at 3 and 6 weeks of age, the values at 9 and 12 weeks of age were significantly lower in pd/pd males than in pd/+ males. A marked difference was not noted in relative weight changes of the seminal vesicles and prostate between the pd/pd and pd/+ males. Histological examination revealed defects in spermatogenesis in both descended and undescended testes in pd/pd males. The changes first appeared at 9 weeks of age and included vacuolation of nuclei of spermatocytes and spermatids, degeneration of spermatids, and occasionally multinucleated giant cells in the seminiferous tubules. In accessory sexual organs such as the epididymides, seminal vesicles, and prostate, no histological abnormalities were detected. These findings indicate that the substantial cause of infertility in pd/pd males is a disorder of spermatogenesis through which functionally normal spermatozoa are not produced.

In vivo anticlastogenic and antimutagenic effects of tannic acid in mice.

The anticlastogenic effect of tannic acid was studied in vivo in the mouse micronucleus test. The frequencies of micronuclei induced by mitomycin C, ethyl nitrosourea (ENU) or 4-nitroquinoline 1-oxide in mouse bone marrow cells were decreased by the oral administration of tannic acid 6 h before the mutagen injection. The observed suppressing effect was not a reflection of a delay in the formation of micronuclei by the cytotoxic effect of tannic acid. The antimutagenic effect of tannic acid was also investigated in vivo in the mouse spot test using male PW and female C57BL/10 mice. Tannic acid was given orally to pregnant females 6 h before the intraperitoneal injection of ENU on the 10th day of pregnancy. The frequency of pups with recessive color spots induced by ENU was decreased by the administration of tannic acid. The observed decrease was not due to toxic effects on the embryo. These results indicate that tannic acid acts as an anticlastogen and antimutagen in vivo.