Mikko Unkila

Exposure to 2,3,7,8-tetrachlorodibenzo-para-dioxin leads to defective dentin formation and pulpal perforation in rat incisor tooth

A single dose of 1000 micrograms 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg body weight was given to TCDD-resistant (Han/Wistar) young adult male rats. Changes in the skulls and the continuously erupting incisor teeth were evaluated 16 weeks after the administration of TCDD. The skulls of the experimental rats (N = 11) were significantly smaller than those of the control rats (N = 11), and the upper and lower incisor teeth of all experimental rats were significantly thinner than the control rat teeth. The pulps of the lower incisors of all experimental rats were lingually exposed to the oral cavity at their incisal ends. Also in 3 cases the pulps of the upper incisors were exposed, but never in the control rats. Whereas the labial surfaces of the incisors of the control rats were brown, those of the experimental rat teeth appeared greyish and mottled. Histological examination revealed that the pulp chambers in the incisal halves of the affected teeth were larger than normal, at the expense of the thickness of dentin. Towards the incisal tooth ends, odontoblasts gradually lost their polarity and the pulp tissue became necrotic. A dentin zone next to the pulp chambers was irregular. Lingual tapering of the teeth was pronounced, which gave them a mesiodistally flattened appearance. The superficial zone of the otherwise regular enamel was poorly pigmented. In conclusion, a single injection of TCDD was shown to impair normal growth of the skull and incisor tooth formation in rats. The small size of the incisors, their aberrant shape and the defective dentin (and enamel) formation could be mediated by vitamin A metabolism, known to be interfered with by TCDD.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on liver phosphoenolpyruvate carboxykinase (PEPCK) activity, glucose homeostasis and plasma amino acid concentrations in the most TCDD-susceptible and the most TCDD-resistant rat strains.

Abstract Reduced gluconeogenesis due to decreased activity of key gluconeogenic enzymes in liver, together with feed refusal, has been suggested to play an important role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced lethality in rats. This study was carried out to further analyse the toxicological significance of reduced gluconeogenesis by comparing dose-responses and time-courses of effects of TCDD on the activity of phosphoenolpyruvate carboxykinase (PEPCK) in liver, liver glycogen concentration as well as plasma concentrations of glucose and amino acids in both genders of TCDD-sensitive Long-Evans (L-E) rats and TCDD-resistant Han/Wistar (H/W) rats. A dose-dependent decrease in PEPCK activity was observed in H/W rats, but in L-E rats the activity was not decreased. However, TCDD impaired the strong increase in liver PEPCK activity observed in pair-fed controls of the L-E strain. Liver glycogen concentrations were severely decreased in L-E rats and moderately in H/W rats. This effect seems to be secondary to reduced feed intake, since a similar decrease was seen in pair-fed controls. Decreases in plasma glucose concentrations were also more profound in L-E rats than in H/W rats, but pair-fed controls were generally less affected. Circulating concentrations of amino acids were markedly increased in TCDD-treated L-E rats, which is likely to reflect increased mobilization of amino acids and their decreased metabolism in liver. Reduction of liver PEPCK activity cannot account for the sensitivity difference of these two strains of rats in terms of mortality. Nevertheless, the response of both strains of TCDD-treated rats regarding gluconeogenesis is different from that seen in pair-fed controls and suggesting that impairment of this pathway contributes to the development of the wasting syndrome.

Biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds on the central nervous system

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds are an important class of environmental contaminants which induce several types of biochemical alterations. Their effects have been most thoroughly characterized in the liver, especially regarding the Ah receptor-mediated induction of xenobiotic metabolizing enzymes. The behavioral signs exhibited by animals exposed to TCDD (progressive anorexia and body weight loss) suggest a role for the central nervous system (CNS) in TCDD toxicity. At lethal doses, TCDD affects the metabolism of serotonin, a neurotransmitter able to modulate food intake in the brain. This effect is associated with an elevated concentration of free tryptophan in the plasma. There does not appear to be any major changes in catecholaminergic neurotransmitter systems in TCDD-treated rats. Cytochrome P-450 related enzyme activities are induced by TCDD in the brain. As is the case in the liver, this induction does not correlate with susceptibility to TCDD lethality in rats. The involvement of the CNS in TCDD toxicity is still obscure. Elucidation of this role as well as the mechanism of TCDD-induced wasting may well advance our understanding of the regulation of food intake and body weight.

2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) INDUCED ETHOXYRESORUFIN-O-DEETHYLASE (EROD) AND ALDEHYDE DEHYDROGENASE (ALDH3) ACTIVITIES IN THE BRAIN AND LIVER A COMPARISON BETWEEN THE MOST TCDD-SUSCEPTIBLE AND THE MOST TCDD-RESISTANT RAT STRAIN

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent inducer of ethoxyresorufin O-deethylase (EROD) and aldehyde dehydrogenase (EC 1.2.1.3., ALDH3) enzyme activities in the liver. Little is known about their inducibility by TCDD in the brain, although it may be a target organ for TCDD toxicity. Two strains of rat, Long-Evans (L-E) and Han/Wistar (H/W) exhibit an over 1000-fold difference in their LD50-values for TCDD. The induction of EROD and ALDH3 in discrete brain regions and in the liver of L-E and H/W rats were now compared at 10 days after TCDD exposure to assess the role of these responses in the strain difference. Liver EROD and ALDH3 were maximally induced at 5 micrograms/kg and 50 micrograms/kg, respectively, in both strains. In the brain 50 micrograms/kg TCDD was mostly needed to enhance EROD activity in both strains. The induction occurred especially in olfactory bulbs, but was also seen in the midbrain plus thalamus of both rat strains. The induced EROD activity in the olfactory bulb was almost totally abolished by a monoclonal antibody (Mab) 1-7-1 raised against CYP1A1. ALDH3 activities were increased more dose dependently in olfactory bulbs of H/W than L-E rats. In other brain areas measured, ALDH3 activities were induced more in L-E rats. Kinetic factors did not explain the differential induction of EROD and ALDH3 among discrete brain regions. We conclude that both EROD and ALDH3 are induced in the brain by TCDD although the activities are much lower than in the liver. The induction in the brain is region specific with olfactory bulbs being the most responsive area. As in the liver, the TCDD-induced activity of EROD in the brain is primarily associated with CYP1A1. According to the present findings, enzyme induction in the brain does not seem to have a crucial role in determining the strain susceptibility to the acute lethality of TCDD.

2,3,7,8-tetrachlorodibenzo-p-dioxin-induced anorexia and wasting syndrome in rats: aggravation after ventromedial hypothalamic lesion

Long-term regulation of body weight and food intake were studied after rats were subjected to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which causes hypophagia and body weight loss, and to ventromedial hypothalamic lesion, which causes hyperphagia, metabolic changes and obesity. These two factors appeared to have an interaction, as ventromedial hypothalamic lesion initially aggravated the effects of TCDD on body weight and food intake. This was seen in both TCDD-resistant and TCDD-susceptible rat strains. In contrast, if TCDD was given several weeks before the lesion and body weight had stabilized to a low level, no aggravation was seen, but TCDD completely blocked the effects of ventromedial hypothalamic lesion. Thus, TCDD seems to affect the same regulation chain that is involved in the lesioning of the ventromedial hypothalamus. TCDD might serve as a tool in studying different mechanisms of long-term food intake and body weight regulation.

Toxic equivalency factors do not predict the acute toxicities of dioxins in rats.

Risk evaluation of complex environmental mixtures of polychlorinated dibenzo-p-dioxins and related halogenated aromatic hydrocarbons (polychlorinated dibenzofurans, azo- and azoxybenzenes, naphthalenes and some of the biphenyls) is currently carried out by measuring the concentration of each congener in the mixture and then multiplying every figure by its specific constant, toxic equivalency factor (TEF). All congeners are thought to produce highly similar effects albeit at different doses, and the TEFs are believed to represent the potencies of the congeners relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), considered the most toxic derivative of this class of environmental contaminants. Here we compared the acute toxicities of TCDD, 1,2,3,7,8-penta-, 1,2,3,4,7,8-hexa- and 1,2,3,4,6,7,8-heptachloro-dibenzo-p-dioxin in the most TCDD-susceptible (Long-Evans Turku AB; L-E) and the most TCDD-resistant (Han/Wistar kuopio; H/W) rat strain. While L-E rats exhibited the expected rank order of sensitivities to the four dioxins, the higher chlorinated dioxins were more toxic than TCDD (in terms of acute lethality) to H/W rats, with the hexachlorodioxin showing the greatest potency. Even if the doses were adjusted according to the LD50 values, both biochemical and morphological effects elicited by the dioxins turned out to depend, often critically, on strain, congener or the interaction of these two determinants. These findings demonstrate that the dioxins have distinct profiles of acute toxicities and underscore the importance of response and test organism in defining the TEFs.

Mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces circulating melatonin levels in the rat☆

We have previously shown that the prototype for halogenated aromatic hydrocarbons, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), diminishes serum melatonin concentration at the same dose in both the most TCDD-susceptible (Long-Evans, Turku AB; L-E) and the most TCDD-resistant (Han/Wistar, Kuopio; H/W) rat strain. The change developed within 24 h and persisted for at least 28 days after TCDD exposure; was independent of the time of day and was not associated with any morphological damage to the pineal gland. In the present study, we investigated the mechanism of this endocrine effect. Despite a 40-50% decrease in circulating melatonin levels, the pineal content of melatonin, serotonin and 5-hydroxyindole acetic acid remained unaltered and the rate-limiting enzyme of pineal melatonin biosynthesis, N-acetyltransferase, displayed only a relatively minor suppression in activity (30%) in TCDD-treated L-E rats. Likewise, TSDD did not influence the ability of pineal glands from L-E rats to synthesize and secrete melatonin in ex vivo or in vitro experiments. TCDD accelerated the disappearance of exogenous melatonin from the serum in both rat strains. This enhancement probably did not originate in the liver, because liver perfusion studies revealed that even control rat livers were capable of total melatonin clearance in spite of the fact that the melatonin concentration far exceeded physiological levels. Urine excretion of the normal main metabolite of melatonin, 6-hydroxymelatoninsulfate, was reduced by TCDD treatment in both strains. This was accompanied by an altered HPLC pattern of metabolites, especially in H/W rats. We conclude that TCDD decreases serum melatonin levels in rats by enhancing the peripheral, evidently extrahepatic, metabolism of the hormone.

TCDD-induced hypophagia is not explained by nausea.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent known anorexigens with an unestablished mechanism of action. In the present study, the role of nausea in TCDD-induced hypophagia was assessed by a battery of behavioral (conditioned taste aversion [CTA], kaolin consumption, protein selection), biochemical (plasma oxytocin), and antiemetic drug intervention (trimethobenzamine, metoclopramide) approaches. Moreover, both the most TCDD-susceptible (Long-Evans [L-E]; IP LD50 approximately 10 micrograms/kg) and the most TCDD-resistant (Han/Wistar [H/W]; IP LD50 > 3000 micrograms/kg) rat strains were employed in the experiments. L-E rats were exposed to a lethal dose of TCDD (50 micrograms/kg), whereas H/W rats were treated with high but nonlethal doses (50 or 1000 micrograms/kg). TCDD produced a positive CTA response in H/W rats alone. These animals also increased their kaolin consumption more than L-E rats of either gender after TCDD exposure. TCDD decreased the proportional intake of energy from high-protein diet in female L-E rats, but tended to increase it in male L-E and H/W rats. TCDD did not affect plasma oxytocin concentration by itself, but potentiated the elevation caused by the positive control compound, LiCl, in L-E rats on day 8. Neither antiemetic tested had any detectable influence on TCDD-induced wasting. These findings imply that the degree of nausea elicited by TCDD in the rat depends on strain and gender. However, nausea has only a minor, if at all, causal role in the lethal wasting syndrome characteristic of this compound.

Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced brain serotonin metabolism in the rat

It has previously been shown that a lethal dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases the brain concentrations of serotonin precursor, tryptophan, and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in TCDD-susceptible Long-Evans but not in TCDD-resistant Han/Wistar rats. In the present study, TCDD (50 micrograms/kg; LD100 for Long-Evans and nonlethal for Han/Wistar rats) enhanced de novo biosynthesis of serotonin in the brain of Long-Evans but not Han/Wistar or food-restricted Long-Evans rats 10 days after exposure. Furthermore, TCDD increased the plasma level of free tryptophan in Long-Evans rats alone, which may be causally related to the observed effects of TCDD on brain tryptophan levels. Administration of hemin modified the time course of TCDD-induced anorexia although 10 day cumulative food consumption was not altered. Hemin tended to attenuate TCDD-elicited increases in brain serotonin turnover, whereas a beta-adrenergic blocker, propranolol, did not. In the majority of Long-Evans rats, TCDD inhibited the main tryptophan degrading enzyme in the liver, tryptophan pyrrolase, but the rest exhibited augmented activities; these effects were not altered by hemin. TCDD increased the plasma levels of nonesterified fatty acids in Long-Evans (five-fold) but not in Han/Wistar rats. A slight elevation (two-fold) was also seen in food-restricted Long-Evans rats. It is concluded that TCDD selectively promotes brain serotonin turnover in Long-Evans rats and this acceleration is related to increased plasma levels of free tryptophan. The inhibition of tryptophan catabolism in the liver and elevation of plasma nonesterified fatty acids may contribute to these changes.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on tryptophan and glucose homeostasis in the most TCDD-susceptible and the most TCDD-resistant species, guinea pigs and hamsters

We have previously reported that in rats 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) lethality is associated (although not necessarily causally) with changes in brain serotonin (5-HT) metabolism. In the present study, we have examined whether this holds for other species by comparing the effect of TCDD in the most TCDD-susceptible and the most TCDD-resistant species, guinea pigs and hamsters, respectively. Body weight gain of guinea pigs exposed to TCDD (0.3–2.7 μg/kg) diminished dose dependently, while the effect was marginal in hamsters (900–4600 μg/kg). Brain 5-hydroxyindoleacetic acid (the main metabolite of brain 5-HT), brain tryptophan (the precursor amino acid of 5-HT), and plasma free and total tryptophan were not affected at any dose in guinea pigs. In contrast, 4 days after exposure, the levels of plasma free and total tryptophan were consistently increased in hamsters. These, as well as brain tryptophan, were still elevated 10 days after exposure. TCDD did not affect plasma glucose level in either species. Liver glycogen was decreased in a dose-dependent manner in TCDD-treated guinea pigs as well as in their pair-fed controls on day 10. There was no change in liver glycogen in hamsters. The activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase was only depressed in hamsters by all doses of TCDD. We conclude that changes in tryptophan metabolism or in carbohydrate homeostasis cannot explain the wide interspecies differences in susceptibility to the acute lethality of TCDD, although they may correlate with some aspects of its toxicity in certain species.