Cardiovascular responses in unrestrained WKY rats to inhaled ultrafine carbon particles

Based on epidemiologic observations, the issue of adverse health effects of inhaled ultrafine particles (UFP) is currently under intensive discussion. We therefore examined cardiovascular effects of UFP in a controlled animal exposure on young, healthy WKY rats. Short-term exposure (24 h) to carbon UFPs (38 nm, 180 microg m (-3)), generated by spark discharging, induced a mild but consistent increase in heart rate (18 bpm, 4.8%), which was associated with a significant decrease in heart-rate variability during particle inhalation. The timing and the transient character of these responses point to a particle induced alteration of cardiac autonomic balance, mediated by a pulmonary receptor activation. After 24 h of inhalation exposure, bronchoalveolar lavage revealed significant but low-grade pulmonary inflammation (clean air 1.9% vs. UFPs 6.9% polymorphonuclear cells) and on histopathology sporadic accumulation of particle-laden macrophages was found in the alveolar region. There was no evidence of an inflammation-mediated increase in blood coagulability, as UFP inhalation did not induce any significant changes in plasma fibrinogen or factor VIIa levels and there were no prothrombotic changes in the lung or the heart at both the protein and mRNA level. Histological analysis revealed no signs of cardiac inflammation or cardiomyopathy. This study therefore provides toxicological evidence for UFP-associated pulmonary and cardiac effects in healthy rats. Our findings suggest that the observed changes are mediated by an altered sympatho-vagal balance in response to UFP inhalation, but do not support the concept of an inflammation-mediated prothrombotic state by UFP.     

Differential effects of commercial polybrominated diphenyl ether and polychlorinated biphenyl mixtures on intracellular signaling in rat brain in vitro.

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and have been detected in human blood, adipose tissue, and breast milk. Developmental and long-term exposures to these contaminants may pose a human health risk, especially to children. Previously, we demonstrated that polychlorinated biphenyls (PCBs), which are neurotoxic and structurally similar to PBDEs, perturbed intracellular signaling events, including calcium homeostasis and subsequent events such as protein kinase C (PKC), which are critical for the normal function and development of the nervous system. The objective of the present study was to test whether commercial PBDE mixtures (DE-71, a pentabrominated dipheyl ether mixture, and DE-79, a mostly octabromodiphenyl ether mixture) affected intracellular signaling mechanisms in a similar way to that of PCBs and other organohalogens, as an attempt to understand the common mode of action for these persistent chemicals. PKC translocation was studied by determining (3)H-phorbol ester ((3)H-PDBu) binding in rat cerebellar granule cells, and calcium buffering was determined by measuring (45)Ca(2+) uptake by microsomes and mitochondria isolated from adult male rat brain (frontal cortex, cerebellum, and hippocampus). As seen with PCBs, DE-71 increased PKC translocation and inhibited (45)Ca(2+) uptake by both microsomes and mitochondria in a concentration-dependent manner. The effect of DE-71 on (45)Ca(2+) uptake seems to be similar in all three brain regions. Between the two organelles, DE-71 inhibited mitochondrial (45)Ca(2+) uptake to a greater extent than microsomal (45)Ca(2+) uptake. DE-79 had no effects on either neurochemical event even at 30 mug/ml. Aroclor 1254 altered both events to a greater extent compared to DE-71 on a weight basis. When the results were compared on a molar basis, Aroclor 1254 altered PKC translocation and microsomal (45)CaP(2+) uptake to a greater extent than DE-71, however, Aroclor 1254 and DE-71 equally affected mitochondrial (45)Ca(2+) uptake. These results indicate that PBDEs perturbed intracellular signaling mechanisms in rat brain as do other organohalogen compounds and the efficacy between the commercial PCB and PBDE mixtures seem to vary with different endpoints.     

Polybrominated diphenyl ether (PBDE) effects in rat neuronal cultures: 14C-PBDE accumulation, biological effects, and structure-activity relationships.

Polybrominated diphenyl ethers (PBDEs), widely used as flame-retardants, are now recognized as globally distributed pollutants, and are detected in most environmental and biological samples, including human blood, adipose tissue, and breast milk. Due to their wide use in commercial products and their persistent nature, long-term exposure to PBDEs may pose a human health risk, especially to children. Our previous reports showed that the commercial PBDE mixture, DE-71, affected protein kinase C (PKC) and calcium homeostasis in a similar way to those of a structurally-related polychlorinated biphenyl (PCB) mixture. These intracellular signaling events are associated with neuronal development and learning and memory function. The objectives of the present study were to test whether environmentally relevant PBDE congeners, with different position and number of bromines, affected PKC translocation in cerebellar granule neuronal cultures and compare the potency and efficacy of PBDE congeners with their 14C-accumulation. All the tested PBDE congeners increased 3H-phorbol ester (PDBu) binding, and a significant effect was seen as low as 10 microM. Among the congeners tested, 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) increased 3H-PDBu binding in a concentration-dependent manner and to a greater extent than other congeners. These effects were seen at concentrations and exposure times where no cytotoxicity was observed. The efficacy of PBDE congeners varied with their structural composition, and the effects seen on 3H-PDBu binding with some PBDE congeners are similar to those of PCB congeners. Cerebellar granule neurons accumulated all three PBDE congeners (PBDEs 47, 99, and 153) following exposure. At the lowest concentration (0.67 microM), about 13-18% of the total dose of 14C-PBDE congeners was accumulated by these neurons. There were distinct differences in the pattern of 14C-PBDE accumulation among the PBDE congeners. The 14C-PBDE accumulation, either represented as percent basis or nanomole basis, was much lower for the 30.69 microM PBDE 99 and 10.69-30.69 microM PBDE 153 than at the lower concentrations, which may be due to low solubility of these congeners. The accumulation pattern with PBDE 47 did not vary with concentration. On a nanomole accumulation basis, PBDEs 47, 99, and 153 accumulation was linear with time. While the nanomole accumulation was linear with concentration for PBDE 47, it is nonlinear for PBDEs 99 and 153. The pattern of PBDE accumulation seems to correlate with the effects on PKC translocation, with regression values of 0.773-0.991. These results indicate that PBDEs affected PKC translocation in neurons in a similar way to those of other organohalogens, some PBDE congeners are equally efficacious as the respective PCB congeners, and PBDE accumulation correlated well with PKC translocation, suggesting a common mode of action for this group of chemicals.     

Amiodarone- and desethylamiodarone-induced pulmonary phospholipidosis, inhibition of phospholipases in vivo, and alteration of [14C]amiodarone uptake by perfused lung

Amiodarone (AM) pulmonary phospholipidosis in patients receiving this drug is well recognized. We investigated the in vivo phospholipidosis-inducing potency of AM and its major nonpolar metabolite, desethylamiodarone (DEA), in rats, their ability to inhibit phospholipases, and also the effects on pulmonary uptake of [14C] AM. Fisher-344 male rats (200 to 250 g) were given AM or DEA (100 mg/kg/d orally) for 2, 7, or 21 d. Food consumption and body weight gain were significantly reduced by both AM and DEA treatment. The control rats, therefore, were pair-fed. Both drugs increased the number of cells in lavage during the treatment. Lung/body weight ratio increased after 21 d of treatment in AM rats. Mortality increased to 100% by day 10 in DEA-treated rats, unlike in AM-treated rats, where 20 to 30% of the animals died during this period and thereafter. No further mortality occurred during 21 d of treatment. Levels of phospholipids increased in lavaged lung, alveolar lavage cells, and surfactant material in AM- as well as DEA-treated rats. However, there was no significant difference between the two treatment groups. Phospholipases A and C were measured in lysosomal soluble fractions of lavaged lung and sonicated lung lavage cells. Both drugs exerted inhibitory action on phospholipases in the lavage cells but, to some extent, spared phospholipases in lysosomal plus mitochondrial soluble fraction isolated from lavaged lung with reversibility in enzyme inhibition despite continuous treatment. [14C] AM uptake by perfused lung, lavage cells as well as surfactant supernatant was increased in AM- and DEA-treated rats. Again, increase in pulmonary uptake of [14C]AM was similar in AM- and DEA-treated rats. These results thus suggest: (1) DEA is more toxic to rats than is AM, at the dose level used. The ability to sequester AM and the parameters related to phospholipidosis revealed no significant differences between these two analogs. (2) Both drugs are inhibitors of lavage cell phospholipases and also are inhibitory to lung lysosomal phospholipases to a lesser extent. Recovery of lung phospholipases occurred despite continuous treatment. (3) AM- and DEA-induced phospholipidosis increased the uptake of [14C]AM by perfused lung. (4) The mechanism of AM-induced pulmonary phospholipidosis includes selective in vivo inhibition of phospholipases.     

Symposium Overview: Toxicity of Non-Coplanar PCBs

Research into the mechanism of toxicity of PCBs has focusedon the Ah receptor. However, it is becoming increasingly clearthat certain ortho-chlorine-substituted, non-coplanar PCB congenershaving low affinity for the Ah receptor exhibit important biologicalactivities. Actions of non-coplanar PCB congeners in a varietyof biological systems have been discovered and the mechanismsfor these effects are being elucidated. The objectives of thissymposium are to examine the state of knowledge concerning themechanisms of toxic action of non-coplanar PCBs and to identifysimilarities and differences using a variety of biological systems.Effects to be considered will include: neurotoxicity, estrogenicity,insulin release, neutrophil function, calcium regulation, andrelevant signal trans-duction systems. Finally, the symposiumaddresses the need to consider non-coplanar congeners withinthe context of risk assessment. The use of Ah-receptor bindingand its associated biological effects to assess the total toxicityof PCBs may no longer be defensible because of the actions producedby non-coplanar congeners. This symposium provides documentationfor that conclusion and focuses attention on emerging mechanismsof PCB action that have received relatively little attentionto date. The topics presented should be of interest to toxicologjstsinterested in mechanisms of action, in PCB risk assessment,and in regulatory toxicology.     

Modulation of calmodulin properties by amiodarone and its major metabolite desethylamiodarone.

Long-term amiodarone therapy is invariably associated with some side effects. Although its mechanism of action, as an antiarrhythmic drug is well understood, the side effect profile of amiodarone is not yet established. To determine possible mechanisms, the interaction of amiodarone and its major metabolite desethylamiodarone with calmodulin was investigated, since calmodulin is known to regulate Ca2+ transport, cell proliferation and the enzymes involved in signal transduction and nucleotide metabolism. The interaction between the drugs and calmodulin was studied by monitoring intrinsic tyrosine fluorescence of calmodulin and by using a fluorescent probe, N-phenyl-1-naphthylamine (NPN). 14C-Chlorpromazine displacement studies were conducted to differentiate the specific binding sites. The effect on the biological activity of calmodulin was determined with calmodulin dependent phosphodiesterase and Ca2(+)-ATPase. The dansyl calmodulin was used as fluorescent probe to study the effect of these drugs on complex formation between calmodulin and phosphodiesterase. Both amiodarone and desethylamiodarone decreased tyrosine fluorescence of calmodulin with IC50 of 4.9 and 4.4 microM respectively and these interactions were Ca2(+)-dependent. NPN fluorescence was also affected in a concentration dependent manner. These drugs also displaced bound 14C-chlorpromazine from calmodulin and the effect was biphasic. However, desethylamiodarone was more potent than amiodarone. The binding of 3H-amiodarone to calmodulin was modified by a variety of compounds, one class of compounds decreased and the other increased 3H-amiodarone binding to calmodulin. Only, desethylamiodarone inhibited the phosphodiesterase activation by calmodulin with IC50 of 13.2 microM without changing the basal enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of Calmodulin Properties by Amiodarone and its Major Metabolite Desethylamiodarone*

Abstract: Long-term amiodarone therapy is invariably associated with some side effects. Although its mechanism of action, as an antiarrhythmic drug is well understood, the side effect profile of amiodarone is not yet established. To determine possible mechanisms, the interaction of amiodarone and its major metabolite desethylamiodarone with calmodulin was investigated, since calmodulin is known to regulate Ca²⁺ transport, cell proliferation and the enzymes involved in signal transduction and nucleotide metabolism. The interaction between the drugs and calmodulin was studied by monitoring intrinsic tyrosine fluorescence of calmodulin and by using a fluorescent probe, N-phenyl-1-naphthylamine (NPN). ¹⁴C-Chlorpromazine displacement studies were conducted to differentiate the specific binding sites. The effect on the biological activity of calmodulin was determined with calmodulin dependent phosphodiesterase and Ca²⁺-ATPase. The dansyl calmodulin was used as fluorescent probe to study the effect of these drugs on complex formation between calmodulin and phosphodiesterase. Both amiodarone and desethylamiodarone decreased tyrosine fluorescence of calmodulin with IC50 of 4.9 and 4.4 μM respectively and these interactions were Ca²⁺-dependent. NPN fluorescence was also affected in a concentration dependent manner. These drugs also displaced bound ¹⁴C-chlorpromazine from calmodulin and the effect was biphasic. However, desethylamiodarone was more potent than amiodarone. The binding of ³H-amiodarone to calmodulin was modified by a variety of compounds, one class of compounds decreased and the other increased ³H-amiodarone binding to calmodulin. Only, desethylamiodarone inhibited the phosphodiesterase activation by calmodulin with IC50 of 13.2 μM without changing the basal enzyme. Desethylamiodarone was more potent in altering calmodulin-dependent synaptic membrane Ca²⁺-ATPase activity than amiodarone. Desethylamiodarone also inhibited Ca²⁺-dependent complex formation between dansyl calmodulin and phosphodiesterase more effectively than amiodarone. These data suggest that amiodarone and desethylamiodarone interact with calmodulin at specific sites altering its active conformation and some of the side effects associated with amiodarone therapy may be due to this interaction with calmodulin.     

Diet and nutritional status of adolescent tribal population in nine states of India

Tribal population constitutes about 8% of the total population in India. They are particularly vulnerable to undernutrition, because of their geographical isolation, socio-economic disadvantage and inadequate health facilities. Recognizing the problem, Government of India launched different programmes for their welfare. Adolescence is a significant period of growth and maturation. The nutritional status of adolescent girls, the future mothers, contributes significantly to the nutritional status of the community. Therefore an attempt was made to assess the diet and nutritional status of adolescent population from the different tribal areas of India. The available database collected by National Nutrition Monitoring Bureau (1998-99) was utilized for this purpose. Data on a total of 12,789 adolescents (10-17 yrs) was included for the analysis. Four percent of the adolescent girls were married and less than 1% were either pregnant (0.4%) or lactating (0.7%) at the time of the survey. The mean intake of all the foodstuffs, especially the income elastic foods such as Pulses, Milk & Milk products, Oils & fats and Sugar & Jaggery were lower than the recommended levels of ICMR. The intake of all the foodstuffs except green leafy vegetables was lower than that of their rural counterparts. The intake of all the nutrients were below the recommended level, while that of micronutrients such as iron, vitamin A and riboflavin were grossly inadequate in all the age and sex groups. About 63% of adolescent boys and 42% of girls were undernourished (< 5th BMI age percentiles of NHANES). A significant association between undernutrition and socio-economic parameters like type of family, size of land holding and occupation of head of household was observed. Therefore, there is a need to evolve comprehensive programmes for the overall development of tribal population with special focus on adolescents.     

Acute lung injury from intratracheal exposure to fugitive residual oil fly ash and its constituent metals in normo- and spontaneously hypertensive rats

We have recently shown that the spontaneously hypertensive (SH) rats with underlying cardiovascular disease exhibited greater pulmonary vascular leakage and oxidative stress than healthy normotensive (Wistar Kyoto, WKY) rats after a 3-day inhalation exposure to residual oil fly ash (ROFA) particles (Kodavanti et al., 2000). Since host responsiveness to a 3-day episodic ROFA inhalation could be different from a single acute exposure, we examined ROFA and its constituent metal (vanadium, V; nickel, Ni)-induced lung injury after a single intratracheal (IT) exposure. Male SH and WKY rats (12-13 wk) were IT instilled with either saline or ROFA (0.0, 0.83 or 3.33 mg/kg). The bronchoalveolar lavage fluid (BALF) was analyzed for lung injury markers at 24 and 96 h post-IT. Rats were also IT instilled with 0.0 or 1.5 micromol/kg of either VSO(4) or NiSO(4).6H(2)O in saline (equivalent to a dose of 2-3 mg ROFA), and assessed at 6 and 24 h post-IT. Basal levels of BALF protein, macrophages, and neutrophils, but not lactate dehydrogenase (LDH), were higher in control SH compared to control WKY rats. Lung histology of control SH rats exhibited mild focal alveolitis and perivascular inflammation; these changes were minimal in control WKY rats. ROFA-induced increases in BALF protein, and to a lesser extent in LDH, were greater in SH compared to WKY rats. ROFA IT was associated with the increases in BALF total cells in both strains (SH > WKY). BALF neutrophils increased at 24 h and macrophages at 96 h in a dose-dependent manner (SH > WKY). The increase in BALF neutrophils was largely reversed by 96 h in both rat strains. The V-induced increases in BALF protein and LDH peaked at 6 h post-IT and returned to control by 24 h in WKY rats. In SH rats, BALF protein and LDH were not affected by V. Ni caused BALF protein to increase in both strains at 6 and 24 h; however, the control values at 24 h were high in SH rats, and were not distinguishable from exposed rats. The Ni-induced increase in LDH activity was progressive over a 24-h time period (WKY > SH). The number of macrophages decreased following V and Ni exposure at 6 h, and this decrease was reversed by 24 h in both strains. V caused BALF neutrophils to increase only in WKY rats. The Ni-induced increase in BALF neutrophils was more dramatic and progressive than that of V, but was similar in both strains. Lung histology similarly revealed more severe and persistent edema, perivascular and peribronchiolar inflammation, and hemorrhage in Ni- than in V-exposed rats. This effect of Ni appeared slightly more severe in SH than in WKY rats. In summary, the acute single IT exposure to ROFA resulted in greater pulmonary protein leakage and inflammation in SH rats than in WKY rats. The metallic constituents of ROFA produced these effects in a strain-specific manner such that, at the dose level used, V caused pulmonary injury only in WKY rats, whereas Ni was toxic to both strains.     

In vitro effects of polychlorinated biphenyls and hydroxy metabolites on nitric oxide synthases in rat brain

Nitric oxide synthases (NOS) play a key role in motor activity in the cerebellum, hormonal regulation in the hypothalamus, and long-term potentiation (LTP), learning, and memory processes in the hippocampus. Developmental exposure to polychlorinated biphenyls (PCBs) has been shown to affect psychomotor functions, learning and memory processes, and to inhibit LTP. We hypothesized that PCBs may disrupt the regulation of such neurological functions by altering NOSs. We have studied the in vitro effects of several PCB congeners and some hydroxy PCBs on NOS activity in cytosolic (presumably neuronal NOS [nNOS]) and membrane (presumably endothelial NOS [eNOS]) fractions in different brain regions of young and adult rats. Among the two selected dichloro PCBs, the ortho-PCB, 2,2'-dichlorobiphenyl (DCB), inhibited both cytosolic and membrane NOS activity at low micromolar concentrations (3-10 microM) in the selected brain regions of all age groups while the non-ortho-PCB, 4,4'-DCB, did not. 2,2'-DCB inhibited cytosolic NOS to a greater extent than membrane NOS. Pentachloro-PCBs (PeCBs) and hexachloro-PCBs (HCBs) did not have a significant effect on adult cerebellar cytosolic or membrane NOS. However, mono- and dihydroxy derivatives of HCBs significantly decreased cytosolic NOS (IC50s: 16.33 +/- 0.47 and 33.65 +/- 4.33 microM, respectively) but resulted in a marginal effect on membrane NOS in the cerebellum. Among three adult rat brain regions, the hypothalamic cytosolic NOS was the most sensitive to 2,2'-DCB. Also, cytosolic NOS in the cerebellum and hypothalamus of young rats was less sensitive than in the older rats. In summary, these results indicate that only di-ortho-PCB inhibited both NOS and hydroxy substitution of one or more chlorine molecules significantly increased the potency of both ortho- and non-ortho-HCBs. The selective sensitivity of NOS to dichloro- ortho-PCB and hydroxy metabolites suggests that the inhibition of NOS could play a role in the neuroendocrine effects as well as learning and memory deficits caused by exposure to PCBs.