Elsie M. B. Sorensen

Histopathological, hematological, condition-factor, and organ weight changes associated with selenium accumulation in fish from Belews Lake, North Carolina.

Green sunfish (Lepomis cyanellus) were collected from two study sites in Belews Lake, North Carolina, for assessment of correlations between several biological parameters and bioaccumulation of selenium. The fish had elevated concentrations of selenium in the hepatopancreas (liver) and exhibited histopathological and other manifestations of selenium poisoning. Condition-factors of Belews Lake fish were significantly correlated with selenium concentrations in hepatopancreas and skeletal muscle, indicative of capillary permeability changes and resultant edema, supported by the occurrence of significantly increased hepatopancreas-weight-to-body weight ratios in the fish having the higher tissue selenium levels. Gill lamellae were noticeably swollen and vacuolated. Hematocrits of the selenium-contaminated fish were significantly lower than those of the reference fish. The hepatopancreas exhibited lymphocyte infiltration, vacuolation of parenchymal hepatocytes around the central veins, and increased numbers of Kupffer cells. The mesonephros exhibited focal intra-capillary proliferative glomerulonephritis. Hearts showed swollen, inflammatory cell-filled pericardial spaces, diagnosed as possible uremia-induced pericarditis. Ovaries in fish with the higher liver selenium levels exhibited numerous necrotic and ruptured egg follicles. These conditions were observed in the Belews Lake Site 2 fish, but did not occur in reference green sunfish, nor did they occur consistently in fish from the less-contaminated Belews Lake Site 1.

An in vitro approach to the study of target organ toxicity of drugs and chemicals

SummaryA major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.

Selenium accumulation, reproductive status, and histopathological changes in environmentally exposed redear sunfish

Comparisons were made of the accumulation of selenium, histopathological damage, and reproductive status of redear sunfish (Lepomis microlophus) collected in July 1986 from Martin Lake (a contaminated site) and Lake Tyler (a reference site). Hepatic concentrations of selenium were four times higher in Martin Lake sunfish (7.6±0.5 ppm) than in fish from the reference lake (2.1±0.2 ppm). Redears collected from the contaminated lake had lower condition factors than individuals collected from the reference site. Sunfish with elevated levels of hepatic selenium had substantial alterations in the liver including necrosis, cytoplasmic vacuolation, and Kupffer cell proliferation. The ovaries of mature fish collected from Martin Lake frequently had atretic follicles, abnormally shaped follicles, connective tissue hypertrophy, asynchronous oocyte development, and an overall reduction in the number of developing oocytes. These histopathological changes in the ovaries of Martin Lake sunfish were not accompanied by alterations in gonadal steroid titers in the blood. No histopathological lesions could be detected in the testes of Martin Lake fish. Most of the males collected from the contaminated site were immature and had lower circulating levels of sex steroid hormones than reference males. The results show that tissue burdens of selenium have declined by 25% since this sunfish population was sampled last in 1981. Further, the results of this study indicate that the overall health and reproductive status of selenium-contaminated fish collected from Martin Lake is still seriously impaired.

Selenium accumulation and cytotoxicity in teleosts following chronic, environmental exposure

M a r t i n Lake is a 5000 a c r e r e s e r v o i r l o c a t e d in Rusk mad P a n o l a c o u n t i e s in e a s t Texas . F r o m S e p t e m b e r 1978 to May 1979, t h i s l a k e was r e p o r t e d to r e c e i v e aqueous , s e l e n i u m l a d e n e f f l u e n t f rom s y s t e m s used to c o l l e c t f ly ash, s c r u b b e r s ludge , and b o t t o m ash (R.W. L O W E R RE, p e r s o n a l c o m m u n i c a t i o n ) . The absence of n o t e w o r t h y d e t r i m e n t a l p h y s i c o c h e m i c a l p a r a m e t e r s and p e s t i c i d e c o n t a m i n a t i o n in t h e l a k e w a t e r was c o n f i r m e d . Numerous s t ud i e s show t h a t se l en ium is a c c u m u l a t e d to tox ic or l e t h a l l e v e l s in t e l e o s t s p e c i e s (NIELSEN and NIELSEN 1978, S A K U R A Y A M A 1960, ELLIS et al . 1937~ H A L T E R et al. 19809 C A R D W E L L e t al. 1976, A D A M S 1976) and t h a t h i s t o p a t h o l o g i c a l , h e m a t o l o g i c a l , and o t h e r c h a r a c t e r i s t i c changes can r e s u l t f rom s e l e n i u m exposu re (ELLIS e t ai. 1937, H A L T E R et al. 1980, C A R D W E L L et al. 1976). In a c o n t i n u e d e f f o r t to m o n i t o r s e l en ium burdens in c r i t i c a l t i s sues of M a r t i n L a k e f i shes and to e s t a b l i s h b i o l o g i c a l c o r r e l a t e s to t h e s e bu rdens , t h i s s t u d y was u n d e r t a k e n u t i l i z i n g two m e m b e r s of t h e C e n t r a r c h i d a e f a m i l y c o m m o n to T e x a s l a k e s and r e s e r v o i r s . F i sh of the s a m e s p e c i e s were c o l l e c t e d f rom r e f e r e n c e l a k e s for c o m p a r i s o n .

ROLE OF CALCIUM IN CYTOTOXIC INJURY OF CULTURED HEPATOCYTES

The role of calcium in irreversible cell death has been the subject of much controversy recently. Documented in vitro studies from several laboratories have shown that calcium can have paradoxical beneficial or detrimental effects on liver cells exposed to hepatotoxic agents.ld The present study reviews various types of in vitro liver models for the study of hepatotoxins and provides data supporting the idea that calcium protects hepatocytes from cytotoxic injury by a well-known hepatotoxin.

Effects of cadmium and calcium on the fluidity of plasma membranes

The fluidity of plasma membranes was assessed by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a fluorescent probe. The presence of increasing concentrations of calcium (Ca) (0.5-4 mM), cadmium (Cd) (50-500 microM), or both decreased the motional freedom of the fluorescent probe molecules in plasma membranes derived from both human erythrocytes and rat hepatocytes. The effects of Cd were 3-10 times greater than those of Ca. Increasing concentrations of Cd in the presence of Ca increased the anisotropy parameter, which plateaued at lower Cd concentrations. The presence of Ca diminished the overall effects of Cd on these membranes.

Hematological dyscrasia in teleosts chronically exposed to selenium-laden effluent

Lepomis microlophus (redear sunfish) were collected from Martin Lake, an east Texas reservoir, as well as a reference lake 8 km upstream in the same drainage system. Martin Lake received aqueous selenium-laden effluent from electrical generator plant systems used to collect fly ash, scrubber sludge, and bottom ash; the reference lake received no such effluent (Texas Parks and Wildlife Department, unpublished report). Neutron activation analysis was used to monitor selenium levels in the hepatopancreas (i.e., the liver and associated, disseminated exocrine pancreas), which is one of the major organs used in monitoring selenium exposure. Fish collected from areas adjacent to the selenium-discharge sites in Martin Lake accumulated about 19.8 ppm in the hepatopancreas, compared to approximately 8.4 ppm for fish collected further from the discharge site. Reference redear sunfish accumulated normal levels of 1.8 ppm in the hepatopancreas. Hematological abnormalities were most severe for fish collected from areas adjacent to the selenium discharge site. These abnormalities included altered leukocyte distribution with elevated numbers of hemoblasts, reduced hematocrits, lower hemoglobin concentrations, slightly reduced numbers of erythrocytes, 12% and 23% reductions in mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV), respectively, and microcytic, poikilocytic erythrocytes with elevated numbers of nuclear shadows. These data present a striking illustration of a case in which the hematological characteristics of a freshwater teleost were correlated with selenium accumulation in a critical organ following chronic selenium exposure. Hematological data provided a sensitive indicator of the severity of the impact of selenium-laden generator plant effluent on this teleost species.

CALCIUM AMELIORATION OF CADMIUM-INDUCED CYTOTOXICITY IN CULTURED RAT HEPATOCYTES

SummaryParenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium with or without calcium, and processed for scanning electron microscopy. To assess the severity of cadmium-induced changes, exposed hepatocytes were categorized based upon the extent of morphological damage. Differences in surface blebbing, alterations in microvilli, variations in the degree of swelling, and changes in cell shape were used to categorize the severity of cell damage. A double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) of cellular changes was conducted for each exposure time and for each concentration of cadmium in the presence or absence of calcium. Significant decreases occurred in the percent relative volume of normal, flattened cells present in cultures exposed for 30 min to 50 or 100 μM cadmium in the absence of calcium. In contrast, the percent relative volume of severely damaged spherical cells was significantly increased after exposure to solutions containing 50 or 100 μM cadmium and lacking calcium. Percent relative volume of intermediate cells (which were slightly swollen and showed changes in microvillar number) was significantly increased following a 30 min exposure to all cadmium concentrations in the absence of calcium. The examination of hepatocytes exposed for 60 min showed that the degree of cadmium-induced cytotoxicity was more severe in the absence of calcium than was the case for the hepatocyte cultures exposed for 30 min: approximately 30% more spherical cells and 30% fewer flattened cells were present if cultures were exposed in the absence of calcium for 60 min compared to those exposed for 30 min. The degree of blebbing was significantly greater at all cadmium concentrations in the absence of calcium. The presence of calcium, therefore, reduced cadmium-induced cytotoxicity in primary cultures of rat hepatocytes subjected to morphometric analysis after scanning electron microscopy.

A correlation between selenium accumulation in sunfish and changes in condition factor and organ weight

Abstract To monitor the biological impact of chronic, low-level selenium exposure, redear sunfish Lepomis microlophus were collected from various locations in Martin Lake which had received discharges of selenium-containing wastes from an electrical generating station operation. For comparison, reference redear sunfish were sampled from one uncontaminated lake 8 km upstream in the same drainage system. Those fish collected from Martin Lake locations (which were recognized selenium discharge sites) accumulated the highest levels of selenium in various organs and had significantly reduced condition factors and hepatopancreas-weight-to-body-weight ratios. Linear regression analyses of these data indicated that the Martin Lake fish which accumulated the highest levels of selenium in the hepatopancreas had the lowest condition factors and the smallest hepatopancreas-weight-to-body-weight ratios. Such biological changes could result from selenium-induced toxic effects such as anorexia, poor feed-to-grain conversion ratios, and/or stunting.

Relative toxicities of several nonsteroidal antiinflammatory compounds in primary cultures of rat hepatocytes

Primary cultures of intact, functional heptocytes were used to compare the relative toxicity of four nonsteroid antiinflammatory agents (NSAID)--benoxaprofen, orpanoxin, aspirin, and ibuprofen--with that of indomethacin. The relative toxicity of these compounds was evaluated on the basis of the release of lactate dehydrogenase, levels of urea (an indicator of a liver specific function), viability (based on dye exclusion), and morphology after a 12-h exposure to concentrations ranging from 0 to 1000 microM. Evaluation of the data obtained from these three parameters enabled us to rank these compounds from toxic to nontoxic, in decreasing order to toxicity: indomethacin greater than benoxaprofen greater than ibuprofen greater than or equal to aspirin greater than or equal to orpanoxin. Morphological evaluations of hepatocytes exposed to these agents under the same conditions supported the order of ranking for these compounds. Ultrastructurally, cells exposed to the two highest concentrations of indomethacin were severely damaged, as evidenced by marked cellular necrosis, nuclear pleomorphism, margination, swollen mitochondria, reductions in the number of microvilli, smooth endoplasmic reticulum proliferation, and cytoplasmic vacuolation. In comparison, exposure of hepatocytes to the highest dose of orpanoxin resulted only in increased vacuolation, a slight increase in cellular debris, and increased electron density of the cytoplasm of hepatocytes.