Ralph E. Linder

Morphological Changes in Livers of Rats Fed Polychlorinated Biphenyls

Male and female Sherman strain rats were fed polychlorinated biphenyls Aroclor 1260 and Aroclor 1254 at 0, 20, 100, 500 and 1,000 ppm in their diet. Rats received the dietary levels for eight months. Light microscopic changes consisted of hypertrophy of the liver cells, inclusions in the cytoplasm, brown pigment in Kupffer cells, lipid accumulation, and, at the higher dietary levels, adenofibrosis. Ultrastructural changes of the livers of exposed animals consisted of an increase in smooth endoplasmic reticulum and atypical mitochondria. Lipid vacuoles were occasionally surrounded by concentric membranes. The epithelial component of adenofibrosis consisted of goblet cells and cells that resembled the epithelium which lines the bile ducts. In general, the effect of Aroclor 1254 on the liver was more pronounced than that of Aroclor 1260.

The Fungicide Benomyl (Methyl 1 -(Butylcarbamoyl)-2-benzimidazolecarbamate) Causes Testicular Dysfunction by Inducing the Sloughing of Germ Cells and Occlusion of Efferent Ductules

Adult male Sprague-Dawley rats (100 days of age) were given single oral doses of the fungicide benomyl (methyl 1-(butylcarbamoyl)-2- benzimidazolecarbamate) in dosages ranging from 25 to 800 mg/kg body weight. The testis and excurrent ducts were examined at 2 and 70 days posttreatment to determine the chemical effects on spermatogenesis and the epididymis. Primary effects seen at 2 days postexposure were testicular swelling and occlusions of the efferent ductules. Mean testis weight peaked with 400 mg/kg. Premature release of germ cells (sloughing), detected even with the lowest dosage (25 mg/kg), was the most sensitive short-term response to benomyl. Sloughing was found primarily in Stage VII of the spermatogenic cycle at the lower dosages, but at higher dosages sloughing extended into all stages except for Stages VIII-XI. Occlusions of the efferent ductules of the testis were dose-dependent and correlated with testis weight. Sperm and sloughed germ cells were compacted in the ductal lumen of occluded ductules, which were surrounded by two to four layers of polymorphonuclear leukocytes and other inflammatory cells. Although the caput epididymidis contained sloughed germ cells and appeared swollen, evidence of permanent occlusion was not found. The long-term (70 days) effects of benomyl were decreased testis weight (at 400 mg/kg), dose-dependent increases in seminiferous tubular atrophy, and increases in the number of reproductive tracts containing occluded efferent ductules. Fibrosis, granulomas, and abnormal growth were long-term consequences of occluded ductules, which were present 100% of the time in testes containing 26-100% seminiferous tubular atrophy. Only a few testes were found to be completely regressed. Occlusion of efferent ductules and disruption of the seminiferous epithelium by sloughing of germ cells are overlapping dose-dependent mechanisms responsible for short- and long-term effects of benomyl on the rat testis.

The effect of polychlorinated biphenyls on rat reproduction

Abstract Reproduction, pathology and acute toxicity were studied in Sherman rats exposed to the polychlorinated biphenyls, Aroclor 1254 and Aroclor 1260. Rats exposed to Aroclor 1254 at dietary levels of 20 ppm or more had fewer pups per litter than the controls in the F 1b and F 2 generations. The 100 ppm exposure level of Aroclor 1254 increased mortality in the F 1b offspring and markedly decreased mating performance of the F 1b adults. The 500 ppm dietary level of Aroclor 1260 reduced litter size and decreased survival in the F 1 litters. Dietary levels of 5 ppm Aroclor 1254 and 100 ppm Aroclor 1260 had no effect on reproduction in rats exposed through two generations. Liver weights were increased in 21-day-old F 1 male weanlings at the 1 ppm level of Aroclor 1254 and in either sex of F 1 and F 2 weanlings at 5 ppm or higher levels of both Aroclor 1254 and 1260. Histological changes in the liver and increased liver weights were observed in adult rats exposed to the higher levels. Pregnant rats given Aroclor 1254 at the rate of 100 mg/kg/day on days 7–15 of gestation produced grossly normal litters, but only 30·1% of the pups survived to weaning. Reproduction and pup survival were not affected at dosage rates of 50 mg. Aroclor 1254/kg/day or 100 mg Aroclor 1260/kg/day. Oral LD 50 values in 3–4-wk-old male rats were 1295 and 1315 mg/kg for Aroclors 1254 and 1260, respectively. The iv LD 50 for Aroclor 1254 in adult females was 358 mg/kg.

Measurement of Epididymal Sperm Motility as a Test Variable in the Rat

Several environmental contaminants, notably dibromochloropropane (Whorton et al. 1977) and kepone (Taylor et al. 1978; Cannon et al. 1978) have been implicated in sperm deficiencies among occupationally exposed males. These incidents emphasize the need for adequate testing of chemicals for effects on the male reproductive system. Although important in clinical diagnosis, the evaluation of sperm motility has not been used extensively as a tool in chemical toxicology, particularly in commonly used small laboratory species. Measurements of sperm motility parameters are complicated by several variables including sample manipulation, temperature, cell concentration, time factors and the method of quantitation. In the present study, a simple objective procedure to estimate the proportion of motile epididymal spermatozoa in the rat has been developed. Emphasis is given to details of handling the sperm sample to minimize intersample variation due to external factors. Quantitation of progressively motile spermatozoa is achieved simply by standard manual and electronic blood cell counting techniques. The difficulty in visual tracking of multiple motile spermatozoa is circumvented by counting only nonmotile cells. An index of motility is calculated from the difference in the nonmotile and total counts, each made on separate aliquots of a sperm suspension. The procedure described is an elaboration of a technique used by Mason and Thompson, 1977. The modified technique is demonstrated in rats given selected doses of dinoseb (2-sec___-butyl 4,6-dinitrophenol), a testicular toxicant in the rat (Linder et al. 1982) and ~-chlorohydrin (3-chloro-l,2-propanediol) whose antimotility effects have been documented in several species (Jones 1983).

Multi-residue method for the determination of chlorinated phenol metabolites in urine

Electron-capture-gas chromatographic (EC-GC) methods for the determination of chlorinated phenol metabolites of hexachlorobenzene (HCB) and pentachlorophenol (PCP) in urine are presented. After extraction the sample was reacted with diazomethane to produce the methyl ether of each metabolite prior to determination by EC-GC. An acid alumina column was used for cleanup and separation of methylated phenols into groups. Average recoveries of greater than 80% were obtained from urine fortified with known amounts of the phenol metabolites under investigation. A level of 1 ppb1 was established as minimum detection limit for each phenol metabolite. Previously unreported urinary metabolites of HCB and PCP were found as a result of a rat feeding study. Levels of chlorinated phenol residues from (a) human general population and (b) a worker occupationally exposed to PCP are also included.

Optimization of the Hamilton-Thorn Computerized Sperm Motility Analysis System for Use with Rat Spermatozoa in Toxicological Studies

To optimize the Hamilton-Thorn Motility Analyzer (HTM; Hamilton-Thorn Research, Beverly, MA) for use in reproductive toxicology studies with rat spermatozoa, the accuracy and precision of the instrument were assessed under a variety of instrument settings. Videotapes of both fast- and slow-swimming sperm were analyzed repeatedly to obtain data across a range of sperm velocities as might be encountered as a consequence of exposure to reproductive toxicants. Acquisition rates were varied across the HTM menu choices (30, 19, 10, or 7 frames/sec) as were the number of frames analyzed (5 to 20) at each framing rate. For fast-swimming samples (mean straight-line velocity (VSL) approximately 130 microns/sec) generally good agreement between computer-assisted sperm analysis (CASA) and manually obtained data was found for percentage of motile sperm and straight-line velocity; i.e., CASA values were within 10% of manual values for most frame/rate combinations. The accuracy of these measures held true over a wide range of sperm concentrations and percentage motilities. However, CASA measures were less accurate for sperm samples of lower velocities (mean VSL approximately 50 microns/sec and mean VSL approximately 30 microns/sec) in that the velocity of very slow sperm was overestimated (particularly at 30 frames/sec). A soft-ware change (6.5R) and performing analyses at 19 instead of 30 frames/sec improved straight-line accuracy for the slow sperm and enhanced the discrimination between fast (presumably control) and slow (presumably treated) sperm samples. These data show that this motility analyzer could be successfully configured to evaluate rodent sperm samples. The use of such CASA systems in toxicology studies will provide valuable information that may improve human reproductive risk assessment.

Polychlorinated biphenyls: Distribution and storage in body fluids and tissues of Sherman rats

Abstract An analytical method for determination of PCB (Aroclor 1254) in blood, urine, feces, and tissues of Sherman strain rats after prolonged dietary intake (100, 500, and 1000 ppm) is presented. The method involves homogenization and extraction with hexane, cleanup on a silica gel microcolumn and analysis by electron-capture gasliquid chromatography. Recovery data are shown at 0.1, 5, 14, and 100 ppm. Mass spectroscopic characteristics of PCB in tissues are given. Distribution, storage, and excretion rates during feeding and after discontinuance of PCB in the diet are presented.

2,4-Dichlorophenyl-p-Nitrophenyl Ether (TOK)

In a two-generation reproduction study, rats were fed dietary levels of 0, 20, 100, and 500 ppm of 2,4 dichlorophenyl-p-nitrophenyl ether (TOK) (1.8 to 1.1 mg/kg/ day, 9.2 to 5.2 mg/kg/day, and 46 to 26 mg/kg/day, respectively). The survival of the offspring was not affected at the 0 and 20 ppm dietary levels. At the 100 ppm dietary level the survival of the offspring to weaning was reduced and at the 500 ppm dietary level no offspring survived the neonatal period in the two breedings of the first generation. In further studies rats were dosed during pregnancy either by stomach tube or by adding TOK to the diet. The reduction in the number of surviving litters was caused by poorly developed lungs in the exposed groups. Technical TOK had the same effect as 99% pure TOK. Furthermore, 2,7-dichlorodibenzodioxin, which may be a contaminant of TOK, does not affect the maturation process of the lungs when given at a dose of 0.04 mg/kg/day on days 7 to 15 of gestation.

Adenofibrosis in the Rat Liver: With Persistence of Polychlorinated Biphenyls in Adipose Tissue

Fifty male Sherman strain rats were fed 500 ppm of a polychlorinated biphenyl (PCB) (Aroclor 1254) for six months. Five each were killed zero, one, two, three, four, six, eight, and ten months after exposure to Aroclor had ceased. The livers of these rats were examined by light and electron microscopy. Liver lesions persisted although exposure to PCBs ceased. Ten months after exposure ceased, 1,192 ppm PCBs were still present in the rats’ adipose tissue and 22.65 ppm in the rat livers. Aroclor patterns found in the tissues by electron capture gas chromatography differed from patterns of dietary Aroclors. Mass spectral analysis of liver and adipose tissue revealed three major Aroclor components with masses of 324, 358, and 392. These contained isotopic clusters indicative of the presence of C15, C16, and C17, respectively.

The ultrastructure of the paraquat lung lesion in the rat.

Abstract Adult female Sherman strain rats were fed 500 ppm paraquat (1,1-dimethyl-4,4-bipyridylium dichloride) in their diet. Four poisoned as well as three control rats were sacrificed 1, 3, 5, 7 and 11 wk after onset of exposure. Two well-developed paraquat lung lesions were encountered. The alveoli of these lesions contained a very prominent amorphous material which was highly osmiophilic when examined with the electron microscope. It consisted of concentrically arranged whorls which were surrounded by or next to less electrondense lattice-like material. Additional ultrastructural findings in those and other lungs consisted of edematous and vacuolated membranous pneumocytes (type I) as well as endothelial cells. The granular pneumocytes were increased in 3 of 20 lungs of exposed animals.