An evaluation method for hematological and clinico-biochemical values in aged F 344 rats on chronic toxicity tests such as long-term inhalation studies on the effects of diesel exhaust]

To determine the chronic toxicity of diesel exhaust, 2,150 F 344 rats were made to inhale various dilutions of exhaust under specific pathogen-free conditions for 16 hours a day, 6 days a week, for 30 months. Ten experimental groups, each with 120 male and 95 female 5 week-old rats, were set up and 14 males and 9 females were usually sacrificed for measurement of the 51 hematological and clinico-biochemical parameters at 7, 13, 19, and 25 months of age, respectively. These parameters were also examined in all surviving rats after 30 months of exhaust inhalation. In this experiment, no remarkable change in any of the parameters was observed in the periodic examinations. Therefore, tendencies of change in parameters were examined by comparing histograms for control and experimental groups. For the reference histograms, we used the values obtained from 144 males and 116 females aged 13-31 months. The first histogram was based on data from 1,352 rats, and the range from the minimum to the maximum value was divided into eight parts. When almost all the values were centered on a peak in the first histogram, this peak was divided into a further eight parts in the second histogram. Histogram evaluation clarified the differences between the control and experimental groups better than general statistical methods such as Student's t-test.     

Adult offspring long-term effects of high salt and water intake during pregnancy

Offspring from dams subjected to hypereninemia, hyperdipsia, and natriophilia by partial aortic ligation (PAL) showed a long-term modification of their ingestive behavior. These rats, upon reaching adulthood, showed an increased appetite for low-concentration saline solutions (0.1 M) when compared to control rats. They also presented a high intake of a medium concentration NaCl solution (0.45 M) after having been offered a very aversive highly concentrated NaCl solution (1.0 M) along with water for 2 days. An increase was also observed in their salt/water intake ratio following two different thirst challenges, 24-h fluid deprivation or sodium depletion by furosemide treatment. The demonstration of the long-term effect of pregnancy history on salt preference in adult offspring draws attention to the possible physiopathological aspects that may be of relevance, considering the well-established relationship between salt intake and hypertension, a disease most commonly occurring in the adult and aged population.     

Effect of long-term alcohol intake on the cardiovascular system of the rat

A group of rats was fed on control liquid diet, while another group was fed on liquid diet containing alcohol up to 36% of the total calories. After 4, 8 and 12 weeks of treatment ECG, haematocrit, histological structure of the heart, blood pressure, cardiac output, distribution of the organ fraction of cardiac output (by Sapirstein's method and 85Sr labelled microsphere technique), nutritive blood flow and circulatory resistance of the organs were studied. A mild repolarization disturbance was shown by the ECG record of the alcohol exposed animals. Haematocrit values and the histological structure of the heart did not change in any of the groups. Relative heart weight increased, blood pressure, total peripheral resistance and nutritive blood flow of the myocardium decreased, while myocardial vascular resistance increased. There was no significant interaction between the effects of alcohol and the duration of exposure to alcohol for any of the parameters monitored. It is concluded that chronic alcohol intake should be taken into consideration in aetiology of ischaemic heart disease.     

Effect of long-term alcohol intake on the innervation of the rat myocardium

The myocardium of rats kept on DeCarli and Lieber's liquid diet containing alcohol to 36% of the total calories for 8 weeks were examined with Falck-Hillarp's method for the histologic demonstration of catecholamines. Relative fluorescence intensity of the myocardial cells and nerves was measured with the Zeiss-Fluoval microphotometer. Under the effect of alcohol, density of the noradrenergic innervation (number of visualized nerve fibres) decreases and fluorescence of myocardial cells is intensified. The increased fluorescence of myocardial cells can be eliminated with reserpine. It is assumed that chronic alcohol intake induces continuous catecholamine release from monoaminergic nerve fibres of the heart.     

Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation

The induction of inherited DNA sequence mutations arising in the germline (i.e., sperm or egg) of mice exposed in utero to diesel exhaust particles (DEPs) via maternal inhalation compared to unexposed controls was investigated in this study. Previous work has shown that particulate air pollutants (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development (in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates of spontaneous mutation, making this endpoint a valuable tool for studying inherited mutation and genomic instability. In the present study, pregnant C57Bl/6 mice were exposed to 19mg/m(3) DEP from gestational day 7 through 19, alongside air exposed controls. Male and female F1 offspring were raised to maturity and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females. In contrast, a statistically significant increase in the mutation frequency of male mice exposed in utero to DEP was observed (2-fold; Fisher's exact p<0.05). Thus, maternal exposure to DEP results in increased mutation in sperm during development.     

Naloxone blocks exercise-stimulated water intake in the rat

To examine whether opiate receptors modulate exercise-induced water intake, we measured water intake during four consecutive hours after a one-hour swim stress in male, Sprague-Dawley rats. Increased cumulative water intake was found four hours following exercise and this response was naloxone-reversible (P = 0.06). Suppression of water intake in the naloxone-treated, exercised group was most marked in the first two hours after exercise (P < 0.05). Non-exercised rats consumed water at a constant, linear rate (P < 0.05) whether treated with naloxone or saline. These results indicate an endogenous opioid role in regulating exercise-induced water intake in the rat, but do not delineate whether this role reflects a non-specific stress behavior or specific physiological processes related to thirst.     

Interaction of diesel exhaust particles with human, rat and mouse erythrocytes in vitro

Inhaled ultrafine (nano) particles can translocate into the bloodstream and interact with circulatory cells causing systemic and cardiovascular events. To gain more insight into this potential mechanism, we studied the interaction of diesel exhaust particles (DEP) with human, rat and mouse erythrocytes in vitro. Incubation of erythrocytes with DEP (1, 10 or 100 μg/ml) for 30 min caused the highest hemolytic effect (up to 38%) in rats, compared to small but significant hemolysis in mice (up to 2.5%) and humans (up to 0.7%). Transmission electron microscopy of erythrocytes revealed the presence of variable degrees of ultrafine (nano)-sized aggregates of DEP either internalized and/or adsorbed onto the erythrocytes in the three species. A significant amount of DEP was found in rat and mouse (but not human) erythrocytes. Lipid erythrocyte susceptibility to in vitro peroxidation measured by malondialdehyde showed a significant and dose-dependent increase in erythrocytes of rats, but not humans or mice. Unlike in human erythrocytes, total antioxidant status (TAS) and superoxide dismutase (SOD) activity in rats were significantly and dose- dependently decreased. In mouse erythrocytes, DEP caused a decreased in SOD (at 10 μg/ml) and TAS (at 100 μg/ml) activities. In conclusion, DEP caused species-dependent erythrocyte hemolysis and oxidative stress, and were either taken up and/or adsorbed onto the red blood cells. Rat (and to a lesser degree mouse) erythrocytes were susceptible to DEP. Human erythrocytes showed the highest resistance to the observed effects. These species difference should be noted when using rats and mice blood as models for humans.     

Differential effects of water and saline intake on water deprivation-induced c-Fos staining in the rat

We studied c-Fos staining in adult male rats after 48 h of water deprivation and after 46 h of water deprivation with 2 h of access to water or physiological saline. Controls were allowed ad libitum access to water and physiological saline. For immunocytochemistry, anesthetized rats were perfused with a commercially available antibody for c-Fos. Dehydration significantly increased plasma vasopressin (AVP), osmolality, plasma renin activity (PRA), hematocrit, and sodium concentration and decreased urinary volume. Fos staining was significantly increased in the median preoptic nucleus, organum vasculosum of the lamina terminalis, supraoptic nucleus (SON), and magnocellular and parvocellular paraventricular nucleus (PVN), as well as the area postrema, nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVL). Rehydration with water significantly decreased AVP levels and Fos staining in the SON, PVN, and RVL and significantly increased Fos expression in the perinuclear zone of the SON, NTS, and parabrachial nucleus. Rehydration with water was associated with decreased urinary sodium concentration and hypotonicity, and hematocrit and PRA were comparable to levels seen after dehydration. After rehydration with saline, plasma osmolality, hematocrit, and PRA were not different from control, but plasma AVP and urinary sodium concentration were increased. In the SON, Fos staining was significantly increased, with a great percentage of the Fos cells also stained for oxytocin compared with water deprivation. Changes in Fos staining were also observed in the NTS, RVL, parabrachial nucleus, and PVN. Rehydration with water or saline produces differential effects on plasma AVP, Fos staining, and sodium concentration.     

Estrogenic activities of nitrophenols in diesel exhaust particles

We recently isolated 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) from diesel exhaust particles (DEP) and identified them as vasodilators. Because these compounds are alkylphenolic derivatives that might mimic hormones, we evaluated their estrogenic activity by using recombinant yeast screens, myometrial contractility assays, and in vivo uterotrophic assays. Recombinant yeast screen assays showed that both PNMC and PNMPP possess estrogenic activity. Furthermore, ovariectomized 25-day-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 days showed significant increases in uterine weight among those receiving 100 mg/kg PNMC and 0.1 and 1.0 mg/kg PNMPP. To clarify further the estrogenic activity of PNMC and PNMPP, rat uterine horns were monitored in organ bath chambers for myometrial contractility in response to oxytocin (OT). Significant differences occurred in the initial and maximum contractilities to OT at 0.25 and 25 mIU/ml in uterine horns obtained from animals treated with 100 mg/kg PNMC and in the maximum contractilities to OT at 0.025, 0.25, and 25 mIU/ml in those from rats treated with 0.1 mg/kg PNMPP. These results clearly demonstrated that PNMC and PNMPP in DEP have estrogenic activity both in vitro and in vivo and might therefore be considered as endocrine-disrupting chemicals.     

Influences on water intake in the rat after lesions of the septal subareas

It has been suggested that the septum plays an inhibitory role in the behavioral function. Recent work has shown that the septum is heterogeneous from the neuroanatomical perspective. The present study examined the water intake of rats lesioned with kainic acid (0.5 microg/0.5 microl/site) on three septal subregions: anterior medial (MSa), posterior medial (MSp), and lateral (LS) sites. Drinking volume was enhanced mostly in rats with the MSp lesion, and so was locomotor activity. However, these two measures were not significantly correlated. This polydipsia induced by MSp lesion was also found in a chronic domain. Another experiment further determined the dipsogenic effects of polyethylene glycol (PEG; 20%) and hypertonic saline (1 M NaCl) in MSp lesioned rats. Water intake increased significantly after administration of the hypertonic saline treatment, but not after injection of PEG. However, this disparity approached a nonsignificant level 8 hr after thirst challenges were conducted. In addition to revealing septal hyperdipsia. these data suggest that the septal subareas can be functionally heterogeneous in drinking behavior. The dipsogenic response profiles for the cellular and extracellular thirst challenges could be differentially affected by kainate lesion in the MSp.     

Changes in the level of specific proteins in the rat kidney during long-term dehydration]

A significant amount of specific proteins are involved in kidney's response to vasopressin. A relative content of 120 kDa protein in the Wistar rat kidney medulla following a 3-day dehydration, was found to be 0.975 +/- 0.037 and after drinking 4% sucrose solution alone--0.871 +/- 0.038. The difference of protein 120 kDa content was not revealed in Brattleboro rats: in the control rats it was 0.814 +/- 0.044, and in the dehydrated ones--0.854 +/- 0.020. The findings suggest that vasopressin directly regulates the 120 kDa protein level in the kidney inner medulla.     

Effect of long-term thyroxine administration on the endocrine epithelium of rat pancreas]

The effect of prolonged thyroxine administration (0.001 mg/g BW) on pancreatic islets has been studied on 64 Wistar male rats by means of radioautographic, morphometric and electron microscopic methods. The phase response in the amount of the DNA-synthesising cells of the middle class islets has been revealed: the initial increase (5 days) is followed by a decrease (30 days) and then by a return to the control levels (60 days). The level of metabolism in sulphur-containing proteins has decreased in both A- and B-cells. After 30 days of the experiment, B/A cell volume ratio has been shown to increase. Electron microscopic studies have revealed ultrastructural reorganization of B-cells from "resting" B-cells into "dark" B-cells at increased excretion of secretory material.