Biomedical subjects
Publications and source records attributed to G Ugazio.
The biological activity of polysaccharides from the mycelia of 40 Basidiomycetes was studied using an uncommon toxicity test technique, the planaria bioassay, and the better known potato disk bioassay. The results showed the utility of this duo of 'in vitro' tests as a preliminary screening of the toxicity of substances that are present in aqueous fungal extracts.
As a preliminary in vivo approach in order to study the mechanism of toxicity of the veterinary anticoccidial monensin, male Wistar rats were orally administered 0, 2 and 12 mg kg-1 body wt. day-1 of monensin for 7 days. At the end of the experiment, effects of the ionophore on serum creatine kinase, lactic dehydrogenase and selected drug metabolising enzyme activities were investigated. Furthermore, liver, heart and quadriceps femoris muscle samples were submitted to morphological investigations. Clinical signs or increasing levels of enzymic markers of muscle injury attributable to monensin toxicosis have never been observed in treated animals. As a matter of fact all drug metabolising enzymes activities checked have not shown significant changes, except for a significant decrease of ethoxyresorufin O-deethylase (up to 31%) and aminopyrine N-demethylase (17%) activities. Morphologically, mitochondrial cristae fragmentation and initial formation of 'myelinic sheaths-like' structures have been noticed in heart and muscle fibres. As far as rat study is concerned, these results confirm heart and muscle as target organs of monensin toxicity. In addition, these findings suggest that the inhibition of hepatic biotransformation processes following the i.p. administration of the ionophore, as reported previously by other authors, might reflect unspecific cellular toxic effects rather than a specific enzyme damage.
Paraquat (PQ), a broad spectrum herbicide, produces severe lung inflammation and necrosis resulting in pulmonary fibrosis and respiratory failure. Tachykinins are peptides released by sensory C fibers and have the ability of influencing respiratory functions and cellular proliferation. To examine whether the damage caused by PQ involves tachykinins, rats were depleted in their content of tachykinins by systemic treatment with capsaicin prior to PQ exposure. The animal subjected to this treatment showed a 3-fold higher viability compared to those treated with PQ alone (75 vs 27%). Depletion of reduced glutathione (GSH) is associated with oxidative stress produced by reactive oxygen intermediates during PQ metabolism. This is considered to be critical in the pathogenesis of lung damage by PQ. PQ treatment induced a significant depletion of GSH during the first days and a similar effect was also observed in the group of capsaicin-pretreated rats. Four weeks after PQ treatment the levels of GSH were similar to controls in rat pretreated or not with capsaicin plus PQ. This may indicate that the reduced levels of GSH may be associated to the toxicity observed in the acute phase, but not of importance in the final PQ-induced mortality. Neutral endopeptidase (NEP) is an enzyme considered to be critical in controlling the levels of tachykinins. Exposure of crude membrane preparations of rat lung to PQ resulted in a dose-dependent inhibition of NEP activity. Since NEP inactivation may occur in lung following a PQ exposure in vivo, the results indicate that during PQ intoxication a more sustained activity of tachykinins may be present, producing effects such as cell proliferation, fluid extravasation and bronchoconstriction. In conclusion, this finding supports the hypothesis that neuropeptides released from capsaicin-sensitive nerves could be involved in the modulation of PQ-induced lung damage.
Hepatic bovine microsomes were incubated with Zineb concentrations ranging from 2.5 mM to 2.5 microM. Only the higher concentrations of the fungicide (2.5 and 0.25 mM) elicited a sharp decline in cytochrome P450, cytochrome b5 and total sulphydryl groups content as well as in the activities of NADPH cytochrome c reductase, aminopyrine N-demethylase and aniline 4-hydroxylase. The loss of cytochrome P450 was matched by a concomitant increase in the amount of cytochrome P420, which represents a catalytically inactive form of cytochrome P450. The same concentrations of the fungicide, either alone or in the presence of NADPH 1 mM, failed to increase the amount of thiobarbituric reactive substances with respect to control incubations, thereby excluding the possibility of lipid peroxidation as a contributing factor in the loss of cytochrome P450 and in the inhibition of cytochrome P450-mediated metabolism. It is concluded that Zineb can depress monooxygenase activity in bovine hepatic microsomes mainly through the denaturation of cytochrome P450 and the impaired transfer of reducing equivalents to the complex cytochrome P450-substrate. These mechanisms might also account for the inhibition in lipid peroxidation brought about by the fungicide.
1. We have studied the hepatic microsomal metabolism of ethanol (MEOS), CYP2E1 expression and catalytic activity, and the response to phenobarbital (PB) induction or CCl4 challenge in rats of either sex genetically selected for their preference (P) or aversion (NP) for ethanol. 2. In P versus NP females, the amount of both total cytochrome P450 and P450 binding to metyrapone was lower, whereas the activities of MEOS, aniline 4-hydroxylase (4-AOH), and 4-nitrophenol hydroxylase (PNP-OH) as well as the level of immunodetectable CYP2E1 content were consistently higher. By contrast, no substantial differences were observed between P and NP males. 3. Despite an apparent down-regulation of CYP2E1 expression occurring in all rats as a result of PB induction, P females maintained higher 2E1 levels and showed enhanced MEOS, 4-AOH and PNP-OH activities with respect to NP females. No such changes were detected in the male counterparts. 4. No sex-related differences in CCl4-mediated inhibition of monooxygenase or MEOS activities were evident between P and NP animals. 5. These results indicate that, in females only, the behavioural trait of ethanol preference is apparently associated not only with higher constitutive levels of CYP2E1 and rate of microsomal metabolism of ethanol but also with altered susceptibility to PB induction.
Orthotopic hepatic transplantation is the most diffused surgical treatment used when attempting to substitute an irreversibly damaged liver. However, this practice faces two main problems. Firstly, surgeons need to explant the organ from the donor who is a just deceased human being, with all the implications involved. Secondly, the host patient must be rendered immunologically tolerant to the graft, through pharmacological suppression. Some hepatic alterations allow a structural and functional compensation by means of an autotransplant, but liver cirrhosis, which is widely diffused in humans and easily produced in experimental animals, has not yet been treated with this technique. This research was undertaken with the aim of procuring hepatic cirrhosis in a medium-sized animal--the rabbit--exposed for some months to CCl4 vapours. Preliminary experiments were performed in order to shorten the challenge period by means of liver induction. This approach was unsuccessful because of the natural susceptibility of this species, which was heightened by the barbiturate administration (0.05% sodium phenobarbital in drinking water, for one week). However, induction, rendered impossible the survival after carbon tetrachloride given orally (125 microliters/kg body weight) or by inhalation (1000 ppm for 2 hours). Finally, CCl4 was administered to normal rabbits by inhalation at the initial concentration of 100 ppm, for 2 hours twice a week. The level of the hepatotoxin was subsequently raised stepwise, according to the body weight curve, up to the maximum of 600 ppm by week 23. At the fourth week of intoxication, the metabolic activity of liver microsomes appeared severely depressed, as shown by the 300% increase of hexobarbital sleeping time. The observation of the surface of liver in situ, performed through an explorative laparatomy, showed initial but clear signs of hepatic fibrosis. At sacrifice, after six or ten months of cirrhogenic treatment, the histopathology examination of the liver demonstrated severe and massive cirrhosis, respectively for the two different steps. This experimental schedule appears to be suitable for producing hepatic cirrhosis in a medium-sized animal, which could be used as a model for pioneering attempts of liver autotransplantation. Furthermore, we point out two important aspects of these results. First, the poison has been inhaled by the experimental animals as it generally happens for humans in the environment. Second, the ratio between the length of exposure versus the life span of rabbits--6 months and 8 years, respectively--parallels that reported for human cirrhosis due to halogenated hydrocarbons--5 or 10 years versus 60 or 80 years.
The effects of subchronic administration (90 d) of zineb were studied in male New Zealand White rabbits. Rabbits were allotted to 3 groups of 8 animals each and offered diets containing 0, 0.3 or 0.6% zineb. A marked decline in weight gain, hemoglobin concentration, hematocrit, and erythrocyte and leucocyte counts occurred at the highest zineb dosage. There was a dose-related depression in circulating thyroid hormones, whereas serum lipid concentration, particularly that of cholesterol and triglycerides, increased. Hepatic lipid concentration was considerably reduced in rabbits exposed to 0.6% zineb. Neither serum testosterone nor the activities of selected testicular enzymes showed changes suggestive of testicular involvement. Pathological changes were in agreement with biochemical findings; there was a marked dose-related enlargement of the thyroid showing histological colloid struma. An increase in relative weight and moderate glycogenosis were detected in liver, whereas no lesions occurred in testes. It was concluded that thyroid and liver are the main targets for zineb toxicity in the rabbit. Unlike the results from previous studies conducted on other food-producing species, repeated exposure of rabbits to zineb failed to cause testicular damage. This might be related to the inability of zineb to significantly accumulate in the testes.
Experimental work in our laboratory has confirmed the protective activity of vanadium compounds on hyperglycemia and glycosuria in streptozotocin (STZ) diabetes. Furthermore, diabetic cataract has also been partially prevented. Nevertheless, the combination of a natural antioxidant, vitamin E, with Na3 VO4 has not further enhanced this ameliorating effect. Our experimental approach has been an attempt to block the prooxidant activity of both STZ and vanadate, with the purpose of eliciting the best possible antidiabetic protection. More recently, a lipid soluble synthetic antioxidant U-78517F, a 2-methylaminochroman, has been reported to have a significant protective effect against brain injury and ischemia. This compound inhibits the iron-dependent lipid peroxidation 100 times more effectively than vitamin E. This investigation has introduced a combination of the vanadium compound plus the aforesaid lazaroid, as its (-) enantiomer, U-83836E, in order to improve the insufficient protection when vitamin E was used. For twelve weeks, male Wistar rats, rendered diabetic with STZ, were administered Na3VO4 in drinking water along with the lazaroid carried by the food. Four, eight and twelve weeks after the beginning of the protective treatment, fluid and food intake, diuresis and excreted feces, glycosuria and proteinuria were determined on biological samples obtained in metabolic cages; body weight and glycemia were also recorded. At weeks 6 and 12 of the treatment, the opaqueness of the eye lenses was controlled and registered. At the end of the experiment, circulating glycosylated hemoglobin (HbA1c), fructosamine, N-acetyl-beta-D-glucosaminidase (NAG), and fluorescent peroxides were evaluated. Within the first month of treatment, protection by the combination paralleled that elicited by vanadate alone. At subsequent steps, U-83836E significantly improved the protective effect of vanadate alone on polydipsia and polyuria, but especially on hyperglycemia and glycosuria. The further ameliorating effect of the lazaroid was also observed on HbA1c and NAG, and, most important, on the cataract. In conclusion, these findings demonstrate that the lazaroid U-83836E succeeds in further protecting the most important symptoms of diabetes treated with vanadate, and that this antioxidant acts effectively even when it is administered orally in food, in a non invasive manner.
A single oral dose (430 mg/kg) of atrazine, a widely employed s-triazine herbicide, was administered to young male rats. There was a significant increase of the in vivo elimination of hexobarbital and a significant induction of the activity of 7-pentoxyresorufin-O-dealkylase, while cytochrome P-450 content and other mixed function oxidase activities remained unaltered. The administration of carbon tetrachloride (CCl4) to atrazine pretreated rats did not substantially augment the impairment of drug metabolizing enzymes brought about by CCl4 alone. Results suggest that atrazine behaves like a relatively weak inducer of phenobarbital-inducible families of cytochrome P-450.
The effect of a single dose of carbon tetrachloride (CT), 1,2-dichloroethane (DCE) and the mixture on liver toxicity was investigated. The co-presence of both toxins exerts a more than additive effect on liver necrosis and on TBA-reactive substances produced by liver homogenates incubated at 37 degrees C. Both these effects are prevented in animals treated with vitamin E. The liver GSH is not involved in the synergistic action. The liver triglyceride levels of rats treated with the mixture are lower than in those treated with CT alone. This finding cannot be explained either by an improvement in the lipoprotein secretion or by the fact that the liver receives less NEFA. It is thus likely that the apparent protection against liver steatosis is the sign of more severe damage to the liver cell that partially blocks triglyceride synthesis. The mixture composed of CT+DCE exerts a potentiating action on liver toxicity with CT+DCE having the same characteristics as those, as previously reported, exerted by the co-presence of CT+DBE.
The liver toxicity of several halogen compound mixtures have been tested. The compounds were selected on the basis of their metabolic pathways: carbon tetrachloride (CT) and trichlorobromomethane (TCBM) undergo a dehalogenation via P450-dependent enzyme system, 1,2-dichloroethane (DCE) and 1,2-dibromoethane (DBE) are mainly conjugated with the cytosolic glutathione (GSH) by means of the GSH-S-transferase. The mixture TCBM+DBE shows a more than additive action on lipid peroxidation and liver necrosis. TCBM, like CT, reduces the hepatic level of GSH-S-transferase, increasing the amount of DBE available for cytochrome P450-dependent metabolism, with the production of toxic metabolites. Thus, the behavior of the mixture TCBM+DBE is very similar to that of the mixture CT+DBE, previously reported. Mixtures composed of CT+TCBM and DCE+DBE do not show any synergistic effect on liver toxicity. The results allow one to conclude that the toxicity of mixtures of halogen compounds can be partly predicted on the basis of their metabolic pathways. When the metabolism is quite different, a synergistic toxicity can occur if one pathway interferes with a detoxification mechanism of the other compound. If the two metabolisms are very similar they produce, at most, an additive toxicity.
Previous findings from our laboratory suggested a possible interaction of atrazine, bentazon and molinate with other environmental and/or occupational poisons. The aim of this research was to obtain further toxicological information by using phenobarbital-induced rats and to characterize the effects of these herbicides on the hepatic microsomal metabolism of xenobiotics. Acute experiments have shown that the LD50 is augmented by the barbiturate pretreatment when atrazine is used, remains unchanged in the case of bentazon, but is lowered when molinate is given. Recrystallized atrazine, in the absence of the wetting compounds, elicits the same acute toxicity found when animals are challenged with a commercial preparation. No significant sex-related differences have been observed. In long-term treatment with these toxicants, atrazine shortened the hexobarbital narcosis, but no effect was observed after administration of either bentazon or molinate. Further studies on hexobarbital sleeping time demonstrated that females are more susceptible than males to the narcotic effect of this compound. The induction-like effect of atrazine exposure has been confirmed, mainly in young animals. At the end of the sleeping time, the actual serum concentration of hexobarbital is practically the same, and is not related to the length of the sleeping time. The absence of behavioral alterations in the open field tests exclude possible neurological effects of the triazine herbicide. In conclusion, these data demonstrate that atrazine by itself induces the hepatic pharmacometabolic system, while its metabolites result less toxic than the parent compound. On the contrary, metabolic transformations render the toxic effects of bentazon more severe.
Male and female Wistar rats were administered a diet containing 450 ppm atrazine as early as 60 days prior to the cohabitation period and the same diet was offered to their offspring. Hexobarbital sleeping time and further in vitro assays pointed to a monooxygenase induction which appeared to be more marked in males vs females and most significant in the offspring at weaning. At this age, induction involved also the cytosolic glutathione S-transferase, a phase II enzyme. Results would suggest that the inducing properties of the herbicide can be transferred to the offspring via the placental and/or the mammary route.
The antithrombinic activity of some naturally occurring omega-oxygenated derivatives of ferulenol and ferprenin has been tested. On the basis of these results, both the type of the oxygenated function and the stereochemistry of the terminal double bond seem important for the biological activity.
A mixture of carbon tetrachloride (CCl4) and 1,2-dibromoethane (DBE) results in a significant increase in relative liver weight (RLW) when compared with the effect of each substance individually. At the same time, the liver triglyceride levels of rats treated with the mixture are lower than in those treated with CCl4 alone. This increase in RLW is not caused by simple tissue oedema. Lipoprotein secretion and the concentration of circulating non esterified fatty acids (NEFA) are not involved in the DBE-induced reduction of steatosis provoked by CCl4. The synthesis of triglycerides is significantly depressed in animals treated with the mixture. These findings provide further evidence of the previously observed enhancement of liver toxicity caused by the simultaneous presence of both agents, since the apparent protection against steatosis is due to greater damage to the liver cell, which is no longer able to counter the steatogenic action of CCl4 through a sufficient synthesis of triglycerides.
The toxicity of selenium (Na2SeO3) and mercury (HgCl2) was determined by using a freshwater planarian which is particularly sensitive to pollution, and belongs to a fissiparous breed of Dugesia gonocephala. The mortality and fissiparity frequency of the subjects were studied. They were exposed to intense treatments (48 hours) or for medium to long periods of time (21 days) to either the single compounds or a combination of both, and were fed or fasting. The lethal effect of sodium selenite is correlated to the food intake, whereas the toxicity of mercurous chloride is probably the result of a fixative effect which does not depend on feeding. The 21-day treatment with the first compound has a non-negligible lethal effect which is probably due to an accumulation phenomenon. At doses where an antioxidant effect prevails, fissiparity is stimulated. On the other hand, the second compound reduces reproduction frequency to half the base values. Compared to the Paracentrotus lividus, the Dugesia gonocephala offers various advantages concerning toxicological experiments; besides being easier to handle in the laboratory, it is available all year round and is not subject to seasonal cycles. It is also more susceptible to the toxic effect of mercury, which is a common and highly toxic pollutant, than the sea urchin.
Investigation of the hepatotoxicity of a mixture of two halogen compounds--carbon tetrachloride (CT) and 1,2-dibromoethane (DBE) used in association, especially in agriculture--is reported. Their simultaneous administration in the rat potentiates the necrosis provoked by exposure to CT alone. This damage can be totally prevented by prior treatment with vitamin E, which raises the liver antioxidant level. Determination of the TBA-reacting substances released from the liver homogenates of animals treated with one or both substances also corroborates the importance of lipid peroxidation in the pathogenesis of this potentiation. Furthermore, no significant difference between poisoning by CT and CT + DBE is noted when liver GSH values are measured under the same experimental conditions. This finding rules out the possibility that potentiation is only the simply sum of the damage caused by the two compounds (lipid peroxidation due to a radical initiator for CT, depletion of GSH for DBE). It is thus something more than a mere combination of two mechanisms of action and further investigation is required to unravel its more complex pathogenesis.
The effects of Mancozeb and Zineb, two dithiocarbamate fungicides used to protect vegetables, on rat thyroid and liver function were studied in an acute and a chronic trial. These compounds may be spontaneously or metabolically transformed to ethylene thiourea, a goitrogenic, mutagenic and teratogenic molecule. Sex linked differences in sensitivity and the possibility that toxicity might be potentiated through induction of the microsomal drug metabolising system by phenobarbitone were investigated. When compared with the findings of Ugazio et al. (1985) on the toxicity of ethylene thiourea, the results obtained in this study, bearing in mind the doses employed and the fact that no potentiation by phenobarbitone was observed, suggest that the risks associated with exposure to these two fungicides are less severe than had been supposed.