Sequential histopathologic analysis of alpha-hexachlorocyclohexane-induced hepatic megalocytosis and adenoma formation in the HPB mouse.

A temporal, gross, and histologic analysis of the livers of male HPB black mice maintained on a diet containing 500 ppm alpha-hexachlorocyclohexane (alpha-HCH) was performed at 1, 3, 4, 8, 14, 21, 30, 33, 44, and 50 weeks. Grossly, progressive liver enlargement was first noticed at 3 weeks, hepatic nodules at 21 weeks, and emaciation at 30 weeks. Histopathologic liver alterations included universal hypertrophy of centrolobular hepatocytes first seen at 1 week and the merging of adjacent megalocytic zones at 3 weeks. At 21 weeks, microadenomata and macroadenomata were seen in 2 of 7 mice. At 30 weeks, adenomata occurred in 7 of 8 mice and at 33, 44, and 50 weeks in 6 of 6, 5 of 5, and 5 of 5 mice, respectively. Individual adenomata were composed of large well-packed cells with basophilic and acidophilic pale-staining or lipid-laden cytoplasm forming disorganized cords of variable thickness. Depending on the stage of development, adenomata were classified into 4 subtypes. Subtype I, the earliest form seen, arose within megalocytic areas and was composed of a small number of megalocytic cells exhibiting loss of polarity. Subtype II was smaller than a liver lobule. Subtype III was larger and at times resulted from the merging of adjacent subtype II nodules. Subtype IV included the largest adenomata, most of which resulted from coalescing smaller sized subtypes. Under the conditions of this experiment, neither hepatocellular carcinoma nor metastases in the lungs were detected. It was concluded that if alpha-HCH-induced hepatocellular adenoma is ever to give rise to hepatocellular carcinoma, this transformation must progress very slowly.

A 60-Hz magnetic field increases the incidence of squamous cell carcinomas in mice previously exposed to chemical carcinogens.

The exposure of tumor-bearing SENCAR mice to a magnetic field of 2 mT at a frequency of 60 Hz for 52 weeks, was found to increase the rate of malignant conversion in chemically-induced dorsal skin papillomas. Detailed histopathology revealed the presence of squamous cell papillomas and squamous cell carcinomas in both sham and magnetic field exposed mice at week 52. However, of the nine mice assessed as having squamous cell carcinomas, eight came from the group exposed to magnetic fields, a difference which is statistically significant at P = 0.03.

Effects of the spermicide nonoxynol-9 on the pregnant uterus and the conceptus of rat.

In a series of experiments the embryotoxic potential of nonoxynol-9 (N-9) was investigated in adult female rats given a single per vaginam application of 5 mg/100 g (0.1 ml/100 g) of this spermicide on day 3 (pre-implantation period) or 7 (postimplantation period) of gestation. Control rats were given physiologic saline (0.1 ml/100 g) intravaginally. The vulvar labia were apposed for 24 h by metallic clips to prevent leakage of the solution. Groups of dams treated on pregnancy days 3 and 7 were killed by CO2 inhalation on gestational days 6, 9, 12 and 15, and 8, 9, 10, 12 and 15, resorption and total resorption of the conceptus, embryonal and placental resorption and total resorption of the conceptus, embryonal and placental necrosis, placentitis, endometritis, multicystic endometrium, and diffuse or segmental dilatation of the uterine horns. Generally, the incidence of these lesions varied with the length of time after N-9 was administered and it was consistently higher in the females treated on pregnancy day 3 than in those treated on day 7. Acute vaginitis waned with time after the application of N-9. It was concluded that under the conditions of this study, N-9 is embryocidal/fetocidal in the rat if administered during the first week of gestation. The impairment of embryonal/fetal development was attributed to the N-9-induced changes in the endometrium, the placenta and/or the embryo.

Nerve cell degeneration and progeny survival following ethylenethiourea treatment during pregnancy in rats.

Ethylenethiourea (ETU)-induced early histologic changes in fetal CNS and their effect on postnatal survival was studied at 0, 15 or 30 mg/kg administered as single oral dose on day 13 of pregnancy. Fetuses, from 4-6 dams killed at post-treatment intervals of 12, 24, 48 and 72 h were fixed and studied for histopathological changes following routine methods. The remaining dams were allowed to litter and their progeny was studied for postnatal survival until 80 days of age. Histologic study revealed the presence of karyorrhexis in the germinal layer of basal lamina of CNS extending from the thoracic spinal cord to the telencephalon twelve hours after treatment with 30 mg of ETU/kg. At 48 h post-treatment, the spinal cord showed obliteration and duplication of the central canal and disorganization of germinal and mantle layers. In the brain, the ventricular lining was focally denuded, neuroepithelial cells were arranged in the form of rosettes and the nerve cell proliferation was disorganized. In the 15 mg of ETU/kg group, cellular necrosis was less severe and consisted of degeneration in a single or a small group of cells widely dispersed in the germinal layer of neuraxis. The initial degenerative changes were observed in a specific nerve cell type, identified as the undifferentiated migrating neuroblast. In the postnatal study, since survival was reduced to 50% at the 30 mg/kg and unaffected at the 15 mg/kg, it was concluded that necrosis of neuroblasts up to a certain degree was compatible with postnatal life until adulthood.

Morphologic evidence for vaginal toxicity of Delfen contraceptive cream in the rat.

The morphologic effects of Delfen contraceptive cream on the female genital tract were evaluated in young female rats given a single per vaginum application (0.1 g/100 g) of this spermicidal agent. Control rats received intravaginal instillates of 0.1 ml/100 g of physiological saline. Immediately after dosing, the vulvas of one-half of the treated and control females were shut by metallic clips to prevent leakage of the material. The females were killed 24 h post-treatment. The genital tracts of all control females were unremarkable. Treated females, with or without vulvar labial apposition, developed acute cervico-vaginitis of varied intensity, but as many as half of those without labial apposition failed to develop any pathologic changes. It was concluded that Delfen cream is capable of causing genital tract injury in rats similar to that induced by an aqueous solution of the active ingredient, nonoxynol-9, and that the incidence of this effect is enhanced if leakage of the cream is prevented by application of vulvar clips.

Skin reservoir formation and bioavailability of dermally administered chemicals in hairless guinea pigs.

There is concern as to whether dermally applied chemicals that remain in the skin after exposure are bioavailable and should be included as part of the systemic dose; this study was conducted to investigate the temporal relationship between the skin depot and absorbed dose. Single doses of 14C-labelled phenanthrene, benzo[a]pyrene or di(2-ethylhexyl) phthalate were administered dermally to groups of four female, Hartley hairless guinea pigs which were housed individually in metabolism cages to collect urine and faeces for radioassay. The animals were sacrificed at 6 hr, 24 hr, 48 hr, 7 days or 14 days after dosing to harvest skin specimens for the determination of radioactivity by autoradiographic and liquid scintillation methods, and to determine the dose that remained in the body. It was found that for all three compounds the amount of chemical left in the skin decreased over time while the cumulative percent dose excreted in urine and faeces increased. The autoradiographic results were consistent with those obtained from the liquid scintillation method showing a gradual decrease in radioactivity grain accumulation over the time periods for the three compounds, with the highest grain density observed around hair follicles of the skin. The results of this study indicate that the chemicals left in the skin after surface washing eventually enter the systemic circulation and should be considered as part of the total dose absorbed, and that the hair follicle may play an important role in percutaneous penetration.

Experimental oral toxicity of domoic acid in cynomolgus monkeys (Macaca fascicularis) and rats. Preliminary investigations.

A recent outbreak of marine food poisoning in humans was attributed to the consumption of blue mussels (Mytilus edulis L.) contaminated with domoic acid (DA) that was produced by the diatom Nitzschia pungens. The clinical and morphological effects of single oral doses of extracts of mussels contaminated with DA or of DA isolated from toxic mussels were investigated in small groups (one to six) of cynomolgus monkeys (Macaca fascicularis; 0.5-10 mg DA/kg body weight) and of Sprague-Dawley rats (60 to 80 mg DA/kg body weight). Control animals were either given saline or were not treated. To test whether monosodium glutamate, present in the food consumed by some affected humans, and dimethylsulphoxide, suspected of being present in the plankton, enhanced the response, monosodium glutamate (at 0.25% of mussel extract bolus) or dimethylsulphoxide (at 1 g per bolus) were co-administered to two (one each) of the DA-treated monkeys. DA-treated monkeys developed transient excitation characterized by vomiting. DA-treated rats showed withdrawal followed by hyperexcitation and death (in one case). Mild to moderate central nervous system lesions consistent with neuroexcitation were present in both monkeys and rats. The addition of monosodium glutamate and dimethylsulphoxide had no significant effect on the appearance and severity of central nervous system clinical signs and lesions. The wide variations in the response of test animals to orally administered DA were attributed to the protective effect of vomiting, and to suspected incomplete or slow gastro-intestinal absorption of the toxic agent. The results reinforce the view that DA is an emetic and that under appropriate conditions may also inflict excitotoxic central nervous system damage.

Domoic acid poisoning and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract.

Consumption of cultivated blue mussels from Prince Edward Island was recently associated with episodes of gastro-intestinal and neurological distress. Extracts of the toxic mussels, tested in the mouse bioassay for paralytic shellfish poison, caused an atypical response characterized by scratching, convulsions and death. The present investigation shows that the domoic acid present in toxic mussels can produce in mice and rats signs identical to those induced by mussel extracts. These studies, preliminary in nature by virtue of the scarcity of domoic acid, gave ip no-effect levels in mice of 0.59 mg/kg body weight based on the behavioural response (scratching) and 2.4 mg/kg for death. These levels correspond to levels of 24 and 94 ppm in mussels. When administered orally doses of between 35 and 70 mg domoic acid/kg body weight were required to produce toxicity in mice and rats. This reduced toxicity is consistent with a lack of absorption from the gastro-intestinal tract: faecal excretion accounted for 102 +/- 17% and 98 +/- 12% (mean +/- SE) of the domoic acid administered to mice and rats, respectively. Since human intoxication occurred at an estimated 1-5 mg domoic acid/kg body weight, susceptible individuals appear to be more sensitive than rodents to the oral toxicity of domoic acid.

Neuropathology of excitatory neurotoxins: the domoic acid model.

A novel type of intoxication in Canada in 1987 was traced to consumption of cultivated mussels contaminated with the excitotoxin domoic acid. Studies carried out in rats and monkeys revealed that parenterally administered domoic acid induces in rats neuroexcitatory phenomena culminating in seizures. Monkeys respond with gagging, emesis and less clearly evident seizure activity. CNS damage consisting of dendrotoxic and gliotoxic edema and nerve cell degeneration occurs in structures of the limbic system and the retina in both species. CNS lesion distribution similarities in animals treated with domoic acid or kainic acid suggest that these excitotoxins share a common pathogenesis mediated by glutamic acid, a putative endogenous excitatory neurotransmitter.

Histomorphogenesis of soman-induced encephalocardiomyopathy in Sprague-Dawley rats.

Although myocardial damage caused by soman has been previously reported, its relation to brain damage is unclear. In order to clarify this relationship, we examined the histomorphogenesis of central nervous system (CNS) and myocardial lesions in Sprague-Dawley rats, given atropine methylnitrate (20 mg/kg) and HI-6 (125 mg/kg) ip 10 min before a single injection of 0 or 130 micrograms soman/kg (sc) and sacrificed 45 min and 1.5 hr, 3 hr, 24 hr, and 72 hr later. Bilaterally symmetrical CNS damage began with vacuolation of the neuropil and was followed by astrocytic degeneration and neuronal necrosis culminating in liquefaction necrosis and focal hemorrhage. The cerebral cortex, limbic system, thalamus, and substantia nigra were common target sites. Repair in affected sites was characterized by capillary endothelial cell proliferation, microgliosis, and reversal of microvacuolation. Myocardial damage began with myocytolysis and contraction bands and evolved into coagulative myocytolysis and replacement fibrosis with a transient recruitment of acute inflammatory cells. The left ventricle, especially its free wall and papillary muscles, was consistently affected. There was good correlation among seizures, CNS damage, and myocardial lesions at all times following treatment. The results support the view that CNS lesions are associated with protracted seizure activity and provide evidence that myocardial damage is neurogenic.

Reversal by testosterone of atrophy of accessory genital glands of castrated male sheep. A histologic and morphometric study.

The histologic features of male accessory genital glands of entire sheep (group I), castrated sheep (group II), castrated sheep treated with 40 daily intramuscular injections of 50 milligrams testosterone propionate (group III), and castrated sheep treated with 600 milligrams testosterone propionate 72 hours before death (group IV) were compared. Sheep were castrated at 3 months old and all sheep were killed when 15 months old. Volume fractions of glandular tissue, intralobular fibromuscular tissue and perilobular fibromuscular tissue of the seminal vesicles and Cowper's glands fluctuated significantly (P less than 0.05) during postcastration atrophy and after repeated testosterone treatment. Atrophy in sheep in group II was least in the prostate but greatest in Cowper's glands, seminal vesicles and ampullae of vasa deferentia. Seminal vesicle plexi, whose cytons had a statistically significant (P less than 0.05) degree of shrinkage, also were atrophied. After treatment with testosterone the postcastration atrophy of plexal neurons was almost reversed in sheep in group III. There also was hypertrophy of epithelial cells but the testosterone treatment failed to reduce to normal the fibromuscular volume fraction of the accessory genital glands. Testosterone propionate treatment of sheep in group IV failed to elicit appreciable morphologic changes. These results are compared with our previous findings on the content and uptake of zinc by the accessory genital glands. It is suggested that accumulation of zinc in the accessory genital glands of sheep is not necessarily closely linked to normal histologic appearance.

The pathology of arylmercurial poisoning in swine.

To produce arylmercurial poisoning, phenylmercuric chloride (PMC) was administered daily to 30 healthy five week-old piglets for periods of up to 90 days. The dosage used ranged from 0.19 to 4.56 mg of mercury (Hg)/kg. Levels exceeding 2.28 mg Hg/kg daily were moderately toxic. The disease occurring in this intoxication resulted from injury to the kidneys and large intestine. Fetid diarrhea and failure to gain weight were consistent clinical signs. The primary gross lesions were necrotic typhlitis and colitis, and nephrosis. Degeneration and necrosis were found in affected organs. Regeneration was prominent in the proximal convoluted tubules. The pathology of this disease was similar to that described for mercuric chloride poisoning in other species and, presumably, reflected the ease with which PMC was metabolized to release mercuric ion.Tissue analysis for mercury suggested that only certain target organs, such as kidney and colon, accumulated significantly high levels of mercury. This, presumably, resulted from rapid metabolism of the compound and excretion of mercuric ion in the kidney and colon. The net effect was to spare other tissues, and to injure the excretory organs when the dose level was sufficiently high.

Interface between pathology and epidemiology in carcinogenesis.

The bottom line here is how or whether our toxicological studies relate to ourselves, to human neoplasia--but the latter is, in important ways, imperfectly defined by the best that can so far be arranged using morphological and epidemiologic methods. There are at least three basic questions: 1) with what precision can the histopathologist define and diagnose cancer in humans--hence emphasis here upon human cancer models, 2) to what extents can difficulties be resolved using animal models, where some similar difficulties are met, and 3) to what extents can epidemiology resolve these difficulties. Not all these problems can be resolved, and the investigator is left with a residue of questions for which no answers are presently available. Some of these are exemplified or precipitated by inconclusive or mistaken diagnoses. In the future awaits practical help from specific biochemical markers and morphometry, but in the present, vigilance and consultation should limit error to a small fraction of observations. In the meantime, existing methods and data are perfectly adequate for study and action in various clinical problems, eg. transmission of familial polyposis of colon, definition and control of various adverse drug reactions.

Choline deficiency and the reversibility of renal lesions in rats.

A transient choline deficiency was induced in young rats subsequently fed a nutritionally complete purified diet during recovery periods of 0 to 119 days. The purpose was to investigate the pathomorphology of the renal lesions and relate this to observed biochemical changes. Acute renal lesions consisting of tubular epithelial cell necrosis and tubulorrhexis were observed in rats immediately after the insult. Chronic renal lesions consisting of interstitial nephritis and scarring were observed 28 to 119 days after the insult; these lesions were qualitatively similar at all times but showed a healing trend as the recovery period lengthened. Kidney and liver weights, liver fat concentration, and serum urea nitrogen concentration were higher in treated rats than in control rats at 0 days (no recovery period allowed) but treatment effects at all other times were minor. Significant changes occurred in serum phenylalanine and tyrosine concentrations and in the phenylalanine to tyrosine ratios after recovery periods of 0, 42 and 84 days. It was concluded that the proximal convoluted tubule was most seriously affected and that the chronic lesions represent a potential threat to kidney function in a stress situation. Some implications for human nutrition are discussed.