Subchronic Experiments Research Articles

The effect of dimethoate, dichlorvos, and parathion-methyl on bone marrow cell chromosomes of rats in subchronic experiments in vivo

The three organophosphorous insecticides dimethoate, dichlorvos, and parathion-methyl were investigated in subchronic experiments on bone marrow cell chromosomes. In the literature these compounds were reported to exhibit both positive and negative results in mutagenicity tests demanding further investigations in subchronic tests. The treatment of different groups of male Wistar rats lasted for 6 weeks with 5 treatment days per week at doses of 1 100 , 1 75 , and 1 50 of the LD 50. Following the last treatment, bone marrow cell chromosomes were prepared. The frequency of cells revealing any aberrations as well as numeric and structural aberrations were evaluated. In this test both dimethoate and dichlorvos demonstrated mutagenic effects following subchronic treatment of Wistar rats, while parathion-methyl at doses of 1 100 , 1 75 , and 1 50 of LD 50 displayed no significant mutagenicity.

Recirculating, retrograde heart perfusion according to the Langendorff method for evaluation of MTG--methyl-2-tetradecylglycidate, McNeil 3716--cardiomyopathy.

Recirculating, retrograde heart perfusion according to the Langendorff method was used in an attempt to further elucidate the cardiotoxicity of methyl-2-tetradecylglycidate (McNeil 3716, MTG) and the eccentric hypertrophy elicited by the compound. In subchronic experiments female rats were exposed to MTG 2 x 10 mg/kg and 2 x 25 mg/kg per day for 4 weeks. At various times hearts were perfused ex vivo for up to 2 hr with either 5 mmolar glucose or 0.5 mmolar palmitate as substrate. Substrate uptake (glucose or palmitate) and enzyme release (LDH-lactic dehydrogenase or CPK-creatine-phosphate kinase) were assessed during perfusion. Biochemical analysis (ATP, ADP, AMP, c-AMP, CP, creatine, pyruvate, lactate, glucose-6-phosphate, glycogen, phospholipids, triglycerides and non-esterified fatty acids) were done in hearts before (drug effect) and after perfusion (stress of perfusion). Besides changes in energy metabolism and high-energy phosphate production, as observed in previous experiments (Bachmann et al. 1984) massive changes were seen in energy reserves in heart tissue (ATP, CP, glycogen, phospholipids and triglycerides). As expected, MTG led to significant increases also in non-esterified fatty acids content in hearts.

Tannic acid inhibition of amino acid and sugar absorption by mouse and vole intestine: Tests following acute and subchronic exposure

The acute effects of tannin (tannic acid; TA) on nutrient absorption were studied by measuring sugar and amino acid uptake across the brash border (luminal membrane) of intact intestine in the presence and absence of TA. Incubation of tissue for 4-9 min in TA solution (1 mg/ml) caused a reduction in passive influx ofL-glucose in voles and mice and a reduction in carrier-mediated influx ofD-glucose and total influx ofL-proline in mice, but not voles. In subchronic experiments, mice and voles were fed for 7-14 days a diet with 4% TA, but there was no significant effect on intestinal brush border uptake ofL-glucose,D-glucose, orL-proline (or three other amino acids tested in voles). In a synthesis of our study with others in the literature, three inferences are made from the patterns of effects across solutes, time scales of exposure, and species. First, the transport inhibitory effects following acute exposure are probably mediated by two processes: increased resistance to passive flux across an effective unstirred layer juxtaposed to the brush border membrane, perhaps due to tannin-mucin binding, and reduced Na(+)-coupled nutrient uptake across the intestinal brush border. Second, there is a species sensitivity difference in TA's effect on the second process. Third, the negative effects observed at the acute time scale in vitro do not necessarily occur in animals eating TA subchronically because little TA reaches the luminal membrane, or if it does its effects are quickly reversed when the tissue is removed and washed with solution free of TA.

Effect of a metal mixture in diet on the toxicokinetics and toxicity of cadmium, mercury and manganese in rats.

The purpose of this work was to determine whether a metal mixture added to diet influences the toxicokinetics and toxicity of some elements. The metal mixture (MM) used in these experiments was ash (slag) from a coal gasification plant. The effect of this mixture (5 percent in diet) on the toxicokinetic and on the acute or subchronic toxicity of Cd, Hg, Mn was determined in rats of different ages. Rats were exposed for five weeks in toxicokinetic and six weeks in acute toxicity experiments. Sucklings were exposed through their mothers, which received the MM in diet over the pregnancy and lactation period. In toxicokinetic studies, half of the animals additionally received Cd, Hg or Mn (100, 50 or 2000 ppm, respectively) in drinking water. In subchronic experiments, six-week-old albino rats of both sexes were given MM in the diet and Cd, Hg or Mn in drinking water for 16 weeks. In toxicokinetic studies, 115mCd, 203Hg or 54Mn were administered orally or intraperitoneally to mothers and pups. Exposure to MM had no effect on the absorption, retention and organ distribution of these elements determined six days after radioisotope administration. In acute toxicity studies, exposure to MM in diet had no effect on LD50 values obtained eight days after oral administration of Cd, Hg or Mn to rats of different ages. In subchronic experiments, the effect of individual metals (Cd, Hg or Mn) was similar in animals with and without simultaneous exposure to the metal mixture (only a slight potentiation of a few health-effect parameters of cadmium was noticed in some animals). It is concluded that oral exposure to MM in the diet had almost no effect on the toxicokinetics and toxicity of Cd, Hg and Mn. This could be explained by the low level or low bioavailability of elements from MM, by the metal-metal interaction within the mixture or by the choice of health effect indicators determined. These results are presented as one of the potential approaches for studying the health effect of a metal mixture as occurring in the environment.

Mucopolysaccharidosis and lipidosis in rats treated with tilorone analogues

The experimental immunomodulatory agent tilorone was previously reported to induce generalized mucopolysaccharidosis (lysosomal storage of sulfated glycosaminoglycans) and lipidosis in rats. While lipidosis is a side effect common to many cationic amphiphilic compounds, none of them except tilorone has been known to cause mucopolysaccharidosis in intact animals. The purpose of the present histochemical and ultrastructural study was to examine whether or not mucopolysaccharidosis can be induced by tilorone analogues. Three analogues were selected and administered to rats in short-term and subchronic experiments, and liver, spleen, kidney, and cornea were examined. The analogues caused generalized cellular lesions which had the same histochemical and cytological characteristics and the same distribution as the lesions produced by tilorone. The results show that the ability to induce mucopolysaccharidosis is not a unique property of tilorone; this drug side effect should be taken into account when developing new drugs with molecular structures resembling that of tilorone.

Reversible, hepatic, lysosomal phospholipidosis in rat induced by subchronic daily administration of trospectomycin sulfate

Trospectomycin sulfate is an experimental aminocyclitol antibiotic which has been shown previously to induce the formation of cytoplasmic lamellar bodies in rat and dog liver in subchronic experiments. The effect of repeated daily administration of trospectomycin sulfate on hepatic phospholipid levels and activities of marker enzymes for subcellular organelles was examined. Rats were treated for 30 or 90 days with 0, 50, or 250 mg/kg/day of trospectomycin sulfate prior to being killed, and another group was dosed for 90 days and then allowed to recover for 79 days prior to sacrifice. Transmission electron microscopy showed the presence of lamellar bodies in hepatocytes in both 50 and 250 mg/kg groups at 90 days but no other apparent changes in cellular morphology. Total phospholipids were increased significantly (1.6-fold) only at 90 days (P < 0.01) and only in the 250 mg/kg group. Phosphatidylcholine, phosphatidylinositol, and two acidic lysosomal phospholipids, bis(monoacylglycero)phosphate and acylphosphatidylglycerol, accounted for 42, 35, and 21% of the increase in total phospholipids. Changes in the activities of marker enzymes were generally confined to the 250 mg/kg group at 90 days, with the largest and most significant increases being in the lysosomal enzymes acid phosphatase and hexosaminidase (P < 0.01). Levels of all phospholipids and marker enzymes, with the exception of succinate dehydrogenase, were not significantly different from controls 79 days after cessation of dosing, and lamellar bodies had disappeared. We conclude that repeated trospectomycin sulfate treatment in rat induces a reversible, dose- and time-dependent lysosomal phospholipidosis in liver which is characterized by an increase in lysosomal enzymes and selected anionic phospholipids.

Neurophysiological markers as early signs of organophosphate neurotoxicity

The central and peripheral nervous system effects of acute and subchronic exposure to three organic phosphoro-acid esters (dimethoate, dichlorvos, parathion-methyl) were studied. CNS-dependent variables included mean EEG amplitude, mean frequency of the EEG, and the activity (power density) of six component frequency bands. Peripheral nervous system evaluations included determinations of conduction velocity, and both relative and absolute refractory periods. Cholinesterase activity was measured in blood, brain and other organs. The results indicate that acute large doses of these agents produce substantial changes in these measures of CNS and PNS function. In subchronic experiments it was found that a six weeks administration of 1 50 LD 50 of the chemicals induced early functional changes in both the central and the peripheral nervous systems. It is recommended that when cholinesterase inhibition is detected in humans, functional evaluations of CNS and PNS should follow.

Neurotoxicologic investigations of the pesticide dichlorvos (DDVP). Effects on the central and peripheral nervous system

The neurotoxic effect on the central and peripheral nervous system of dichlorvos (DDVP) was investigated by a computer system in acute and subchronic experiments in CFY male rats. The administered peroral doses were given by gavage; the acute group was given a single 88 mg/kg dose and the 2 subchronic groups were given 1.6 mg/kg or 0.8 mg/kg daily for a period of 6 weeks. Significant changes of the function of CNS - increase of EEG mean frequency, decrease of EEG mean amplitude, that of activity of EEG bands (power density) - and peripheral nervous system - decrease of conduction velocity, increase of relative and absolute refractory periods - were found after treatment with both the single large and repeated small doses of dichlorvos. There were no correlations between the functional disturbances of the central and peripheral nervous systems and the inhibition of the cholinesterase activity in various organs and the blood.

Immuno-, neuro-, and general toxicologic animal studies on a synthetic pyrethroid: Cypermethrin

The toxicologic properties of the synthetic pyrethroid insecticide cypermethrin have been studied in animal experiments. In the course of acute and subchronic experiments performed by the administration of 1 2 and 2 4 , 1 20 , and 1 10 proportions of the oral LD 50 value, no considerable changes were found in the general toxicologic tests. The compound known to affect the central nervous system, however, induced only a mild enhancement of the central excitation level shown by the EEG investigations in the doses applied. In the immunotoxicologic studies an early and dose-dependent suppression was induced as a result of humoral immune response of rabbits immunised by S. typhi following the administration of cypermethrin. The cell-mediated immune response was also decreased. In rats the immune response determined by anti-sheep erythrocyte and antiovalbumin titer as well as by autologous rosette formation of spleen lymphocytes was hindered. The earliest detectable group of symptoms indicating the effect of a mild cypermethrin exposure is the appearance of the positivity of the immunotoxicologic tests. On the basis of the experimental findings the sensitive immunotoxicologic tests have been regarded as of great importance in the series of toxicologic methods.

The effect of clodronate (dichloromethylene diphosphonate) on the mineral concentration of dental enamel and bone of the miniature swine.

Diphosphonates are compounds which prevent the crystallisation of calcium phosphates from solution. Our aim was to study the effects of clodronate (dichloromethylene diphosphonate), one of the diphosphonates, on the mineral concentrations of dental enamel and bone. In a subchronic experiment the effects of this compound were investigated. The drug was administered orally in three doses (100, 300, 1000 mg/kg) per day to 32 Göttingen miniature swines for six months. From the acid etch biopsy of enamel the amount of dissolved hydroxyapatite and the concentrations of fluoride and magnesium were measured. The calcium, phosphorus, fluoride, magnesium, carbonate and citrate concentrations were determined from the cancellous bone sample. All values were calculated for dry bone. Calcium/phosphorus and calcium/magnesium ratios were calculated for both enamel and bone. In tooth enamel the magnesium concentration showed a significant dose dependent decrease and calcium/magnesium ratio a significant dose dependent rise. Calcium phosphorus/ratio exhibited a dose dependent increase in surface enamel. Bone fluoride concentration showed a highly significant and calcium/magnesium ratio a significant dose dependent rise. Clodronate seems to interfere with the remineralization process of tooth enamel, through its effect on magnesium. Incorporation of the drug also appears to exhibit a marked action on the fluoride metabolism of bone. This may require attention in certain long term treatment situations with clodronate in high fluoride areas.

Protective action of ?2-adrenoceptor agonists in hypoxic hypoxia

The experiments on inbred male rats have been carried out to estimate the effect of parenteral vinblastine, an inhibitor of catecholamine axoplasmatic transport, on gastric tumour growth induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) added to drinking water. The maximum tolerance dose of vinblastine was determined in subchronic experiments on 30 rats (0.25 mg/kg subcutaneously, once per week). Chronic experiments with combined introductions of MNNG and vinblastine were performed on 70 rats. Vinblastine was shown to inhibit carcinogenesis on "intestinization" stake and to decrease three-fold the incidence of gastric adenocarcinomas. Vinblastine-induced changes in the behaviour of animals reflect the decreased activity of central adrenergic processes. The role of catecholamines in the mechanisms of specific action of chemical carcinogens is discussed.

Pharmacokinetics and tissue distribution of astemizole in the dog

AbstractThe pharmacokinetics of a single oral dose of 3H‐astemizole (AST) of 1 mg/kg was studied in Beagle dogs and compared with that of a tracer dose superposed at the end of a subchronic experiment. Single and superposed doses were equally well absorbed, and elimination half‐lives of AST and of its metabolites were similar at both occasions (t1/2ß = 3–5 d). Steady‐state was reached after 2–3 weeks of daily dosing. AST and its metabolite desmethylastemizole (DES‐AST) were extensively distributed, mainly to well perfused tissues but only poorly to brain, muscle, and fat. All tissue levels were many times higher than the corresponding plasma concentrations. The superposed tracer dose was well stirred in the tissue compartments and showed instantaneous equilibrium with pretreatment levels. Tissue/plasma level ratios of AST and DES‐AST remained constant with time, indicating that the elimination from tissues proceeded at a similar rate to that from plasma. The extensive distribution of AST and DES‐AST is explained by a strong, but reversible tissue binding that appears to be rate‐limiting for the elimination from the body.

Ethanol induces an increase of harman in the brain and urine of rats.

Harman occurs in rat brain, with the highest concentration in the cerebellum and the lowest in the striatum. 2 g/kg ethanol were ineffective with respect to the concentration of harman in the brain whereas 5 g/kg ethanol caused a time-dependent increase in the cerebral cortex as well as the cerebellum. A toxic dose (8 g/kg) of ethanol elicited no change of harman in the brain 3 h following the application. The rise in the harman concentration in the brain did not correlate with the increase of acetaldehyde in the blood after treatment with ethanol suggesting that several mechanisms are involved in the changes of the levels of harman. In subchronic experiments rats were treated with ethanol over a period of 5 or 6 days. Harman increased in the brain whereby the effect seemed to be more pronounced in the cerebellum than in the cerebral cortex. The concentration tended to increase over time and reached control levels again during withdrawal. The time course of the excretion of harman into the urine was similar to that of the brain in that it increased continuously during the period of ethanol treatment and reached control levels again during withdrawal.

Preclinical toxicology studies with acyclovir: Acute and subchronic tests

Acyclovir (ACV), a new antiherpes drug, was evaluated for toxicity in a series of acute and subchronic toxicity tests. Oral LD 50 values were greater than 10 000 mg/kg in male ICR mice and greater than 20 000 mg/kg in male Long Evans rats. When ACV was given iv, the LD 50 was 405 mg/kg for male mice and greater than 600 mg/kg for male rats. Additionally, LD 50 values for male rats treated sc were 1070, 790, 678, and 650 mg/kg in rats that were respectively, 3, 10, 28 and 71 days old indicating that very young rats were not more sensitive to acute toxic effects of ACV. There were no signs of toxicosis in CD-1 mice given ACV by gavage at dose levels of 50, 150 and 450 mg/kg/day for 1 month. Obstructive nephropathy occurred in rats given 20, 40 and 80 mg/kg/day once each day by rapid iv injection for 3 weeks. Both 5 and 10 mg/kg/day were no effect dose levels. Renal damage caused by precipitation of drug crystals in renal tubules and collecting ducts in rats given ACV by rapid iv injection was readily reversible within 2 weeks. Beagle dogs were given doses of 10, 20, 25, 50 and 100 mg/kg b.i.d. by rapid iv injection for 1 month. All 8 dogs given 100 mg/kg b.i.d. died by the 8th day of treatment; 5 of 8 dogs given 50 mg/kg b.i.d. died after 21 to 31 days of treatment. At 50 and 100 mg/kg b.i.d. the clinical signs of toxicosis were numerous and mainly resulted from the underlying morphological and functional changes associated with hypoplasia of the esophageal and gastrointestinal mucosa, lymphoid tissue, and bone marrow. At the 20 and 25 mg/kg b.i.d. dose levels the kidney was the target organ; the principal indications of altered renal function were increased water intake and hyposthenuria. The dose level of 10 mg/kg b.i.d. was a no effect level for Beagle dogs treated iv. Thus, in subchronic experiments, the rapid iv injection of acyclovir caused precipitation of crystals in the renal tubules, resulting in obstructive nephropathy in rats and dogs. Primary toxicity occurred only in the dog where high doses of acyclovir caused hypoplasia of certain tissues with rapid cell turnover.

The possibility of food contamination with cadmium by means of coloured plastics.

By using spectrophotometric atomic absorption, the release of cadmium from some plastic materials (polyethylenes, PVC, polystyrene) used for manufacturing containers, wraps and household objects (which are coloured with pigments derived from cadmium compounds) was studied. Subchronic experiments (45 days) were carried out in white Wistar rats with extracts from cadmium-containing plastic in comparison with the pigment alone. The excreted cadmium in urine and the activity of some serum enzymes were determined. Histopathological changes of the main internal organs were observed. It was found that some pigments of the Papion type have the property of releasing cadmium in amounts exceeding the admitted sanitary levels, amounts which proved to be toxic, producing enzymic and histopathological changes in kidneys and testes.

Citalopram‐induced Generalized Lipidosis in Rats

This study was focussed on the question of whether or not the potential antidepressant citalopram, which is an amphiphilic cationic compound, can induce generalized lipidosis in animals. In a short term experiment, female rats were treated with a single oral dose (100 mg/kg) and the lymph node was examined by electron microscopy; a significant number of lymphocytes showed lamellated inclusions indicating lipidosis. In a subchronic experiment (7 weeks) female rats received 140 mg/kg per day, and several organs were examined. Marked lipidosis-like alterations were found in lymph node, adrenal cortex and medulla, kidney, lung and in a sympathetic ganglion. Mild lysosomal alterations were found in hepatocytes and retinal pigment epithelium. Lipidosis was very weak, or absent, in retinal ganglion cells, trigeminal ganglion cells and in hypothalamic neurosecretory perikarya. In addition, citalopram produced myopathic alterations in soleus muscle resembling those previously induced with other cationic amphiphilic drugs. Myopathy persisted during a drug-free recovery period of 4 weeks, whereas generalized lipidosis was reversible within 2-4 weeks. The present results support a previously proposed concept concerning the structure-response relationships underlying drug-induced lipidosis.

Acute and subchronic effects of bometolol on blood pressure in hypertensive rats.

Bometolol is a cardiospecific beta-adrenergic blocking drug. The effect on blood pressure was determined in hypertensive rats in acute and subchronic experiments. In acute experiment bometolol, 10-30 mg/kg, p.o., was given to spontaneously hypertensive rats; deoxycorticosterone and salt hypertensive rats; and two kidney, one clip hypertensive rats. Blood pressure was determined continuously for 8 h and after 24 h in unanesthetized condition. The blood pressure lowered by bometolol was dose-dependent in three types of hypertension. In subchronic experiment, bometolol at a dose of 10-30 mg/kg or 100-300 mg/kg, p.o., were given for 5 weeks to deoxycorticosterone and salt hypertensive rats; and two kidney, one clip hypertensive rats. Bometolol did not show antihypertensive effect in either type of hypertension, although bometolol decreased heart rate dose-dependently. Bometolol treatment at the above doses decreased plasma renin activity, heart and kidney weights, and incidence of vascular lesion in either hypertensive rat. Toxic symptom was seen 3 days after bometolol treatment of 300 mg/kg in deoxycorticosterone and salt hypertensive rats.

Toxicity of 5-chloro-3-methyl-catechol to rat: chemical observations and light microscopy of the tissue.

5-chloro-3-methyl-catechol was first time described by GAUNT and EVANS (1961) as a metabolite of MCPA (4-chloro-2-methylphenoxyacetic aci~which is the most widely used herbicide in the Nordic countries. The toxicity of the metabolite 5-chloro-3-methylcatechol bas hot been studied so far. Other catechols like 3,5dichlorocatechol has been shown to be a metabollte of 2,4-D (BOLLAG et ai. 1968a, BOLLAG et ai. 1968b), and it has been proven to be toxic to cells by HORVATH (1971). The catechols are metabolized by bacteria finally to muconic semialdehyde (HORVATH 1970, HORVATH and ALEXANDER 1970). In this work the toxicity of 5-chloro-3-methyl-catechol to maie rats was studied in acute and subchronic experiment in order to relate the histopathological changes in the tissues to the residue levels in the same tissues. A TLC method recently developed by SATTAR et ai. (1977) was used in the clean-up and the quantitative determination of the catechol residues occurred by glass capillary GLC.

Acute and subchronic experiments on the effects of microcrystalline cellulose on various pharmacological and biochemical parameters in mongrel rabbits

The subchronic intravenous application of microcrystalline cellulose (MCC), in a dose of 5 mg/kg twice weekly for 10 weeks, resulted in servere crystal embolism in the lungs of the animals. The clinical parameters were either unchanged (erythrocyte and leucocyte counts, differential blood picture, osmotic resistance of the erythrocytes, total serum bilirubin, serum protein and serum protein fractions) or slightly pathological (hemoglobin content, hematocrit, GOT and GPT). The results from tests of the liver function (by means of bromosulphthaleine clearance) and of the renal function as well as the ophthalmologic examination were not indicative of damaged parenchyma or disturbed microcirculation. Intra-arterial infusions of MCC affected the peripheral muscular blood supply, blood pressure and heart rate.

Experimental investigations of the effect of cigarette smoke exposure on testicular function of Syrian golden hamsters

Changes in the testicles were investigated in 3605 Syrian golden hamsters following chronic and subchronic exposure to cigarette smoke. Experimental results demonstrated that testicular atrophy is age-dependent and partly caused by mechanical factors inherent in the experimental procedure. No differences between experimental and control animals could be observed after chronic smoke exposure. The influence of the age-dependent factor outweighed that of the mechanical factor. In the subchronic experiment, the mechanical factor determined the frequency of testicular atrophy.