F. Coulston

Experimental atherosclerosis in rhesus monkeys: II. Cellular elements of proliferative lesions and possible role of cytoplasmic degeneration in pathogenesis as studied by electron microscopy

Abstract The proliferative (non-necrotic) lesions of experimental atherosclerosis in the rhesus monkeys reported here are similar to those of the human in that they are composed primarily of mature smooth muscle cells, most of them containing lipid. In addition, there were small numbers of primitive cells, fibroblast-like cells, and smooth muscle cells of apparent intermediate maturity. This variety of morphologic classes of cells appears to reflect maturation of smooth muscle cells within the intimal lesion. Accompanying these cells were cells tremendously distended with lipid that could not be identified as to their histogenesis. They were probably either distorted smooth muscle cells or macrophages. Many foci suggesting cellular degeneration were found scattered throughout the proliferative lesions, while only a few were found in stock-fed monkeys. The increased number of cells in the proliferative lesions showing degenerative changes were apparently the indirect or direct result of feeding high-fat diets. Whatever their cause, it is likely that these foci of intracellular degeneration enhanced the development of the intimal lesion by stimulating the cells to proliferate.

Intestinal effects of carrageenans in the rhesus monkey (Macaca mulatta)

Abstract In a study in male and female rhesus monkeys ( Macaca mulatta ), the effects of a native form of carrageenan (HMR), derived from Chondrus crispus , have been compared with those of a degraded form (C16) obtained from Eucheuma spinosum . Both types were administered in drinking-water for 7–14 wk. Monkeys given 1 % HMR (providing an intake of about 1·3 g/kg/day) all gained weight and remained in good condition. Occult blood occurred sporadically in the faeces, as was the case in controls. Only minor changes, none attributable to HMR administration, were found in the intestinal tract at autopsy and on microscopic examination. Similar findings were recorded in animals given 1 % HMR for 11 wk and subsequently, after a recovery period of up to 11 wk on tap-water, given escalating daily doses of 50–1250 mg HMR/kg for up to 12 wk. Monkeys given 0·5 or 1·0% C16 solution gained weight, but in those on 2·0% C16 (an intake of approximately 2·9 g/kg/day) weight losses were considerable. All monkeys on C16 lost blood frequently from the intestinal tract and developed some degree of anaemia. Pathological changes seen in the colon ranged from shallow mucosal erosions to ulceration associated with cellular infiltration, granulation tissue in the lamina propria and formation of multiple crypt abscesses. The severity of these effects was dose-dependent, and some reversal was indicated in monkeys given 2% C16 for 14 wk and then allowed to recover on tap-water for 20–24 wk. A marked difference was thus demonstrated between the effects of degraded (C16) and native (HMR) carrageenan in the monkey.

Experimental atherosclerosis in rhesus monkeys: I. Gross and light microscopy features and lipid values in serum and aorta☆

Abstract Forty-five rhesus monkeys were divided into five groups fed four different high-fat diets and a stock diet. The duration of feeding varied from 33 to 70 weeks. All monkeys fed the high-fat diets developed aortic proliferative lesions composed predominantly of spindle-shaped cells, most of them laden with lipid. The thickest of these proliferative lesions measured 0.85 mm, and most were thinner, suggesting that in the monkey receiving these high-fat diets some proliferative lesions can reach this range of thickness before undergoing necrosis. Atheromatous aortic lesions characterized by necrosis and the accumulation of lipid debris were found only in monkeys receiving high-fat diets for 70 weeks. In monkeys receiving a diet in which the lipid component was 30% peanut oil and 5% cholesterol, the fatty acid composition of the aorta was similar tothat in humans developing coronary artery atherosclerosis, in that the fatty acids showing the greatest accumulation were oleic and linoleic. The largest number of atheromatous lesions, however, was associated with a diet otherwise identical to the peanut oil-cholesterol diet but containing as its lipid component 30% butter and 5% cholesterol. This latter diet also resulted in higher serum lipid levels and higher aortic lipid levels per milligram of DNA than did peanut oil diet. High-fat diets fed to rhesus monkeys appear to produce both proliferative and atheromatous aortic lesions similar to those seen in the human as regards both light microscopy features and lipid composition. Work is now in progress assessing some of the metabolic features of these lesions.

Endocrine effects of chlorinated hydrocarbons in rhesus monkeys

After the rhesus monkey was demonstrated to be a suitable model for man in both metabolic and endocrinological studies, effects of hexachlorobenzene (HCB) and polychlorinated biphenyls (PCB) on the pattern of sexual hormones in cycling female rhesus monkeys were investigated. After confirmed ovulation, four adult female rhesus monkeys were treated during the following cycle with 4 mg/kg/day of HCB, and four other monkeys were treated with the same dose of Clophen A 30. Ovulation was blocked in three PCB-treated and one HCB-treated monkeys. Whereas the levels of luteinizing hormone and follicle-stimulating hormone did not seem to be changed directly by the treatment, low estrogen levels were found during the anovulatory cycles. Studies with PCB- and HCB-treated superovulated rats indicated interaction of the chemicals with ovarian steroidogenesis. Altered hepatic steroid metabolism may also cause low estrogen levels in treated animals.

The response of the hypothalamus to high doses of monosodium glutamate in mice and monkeys: Cytochemistry and ultrastructural study of lysosomal changes

Abstract Administration of high doses (4 or 1 gm/kg) of monosodium- l -glutamate (MSG) in aqueous solution subcutaneously in 100 mice 5–7 days old produced changes in the arcuate nuclei and median eminence of the hypothalamus in 60% of treated mice given 4 gm/kg and in 42% of those receiving 1 gm/kg. The remaining mice appeared to be unaffected. The action of MSG was directed against both neuronal and glial cells. Corresponding doses of MSG given orally to 95 mice elicited a predominantly glial reaction in 28%. When four monkeys were given MSG (4 gm/kg) at 4 days of age, no distinction could be drawn between the appearance of the arcuate nuclei, median eminence, or ependymal cells in treated animals and the same structures in untreated controls. This species difference in susceptibility, as well as individual disparities among the mice, is probably attributable to the dissimilar degree of myelination of the central nervous system present at birth. Cytochemical and ultrastructural studies on the hypothalamus of the affected mice demonstrated a significant increase in the number of lysosomes, with appearance of numerous myeloid bodies. The presumptive intermediate stage of autophagic vacuole formation was not seen. The lysosomal effects observed have served to delineate hitherto unsuspected changes in the cell cytoplasm in affected animals.

Biochemical changes in the liver of mice fed Mirex

Abstract In a study of the hepatic effects of Mirex (dodecachloroctahydro-1,3,4-methano-2H-cyclobuta[cd]pentalene), male Charles River CD-1 mice were fed 1–90 ppm Mirex in the diet for periods ranging from 1 to 70 wk. At regular intervals, heavy mitochondrial, light mitochondrial, microsomal, and soluble fractions were prepared from livers of three to six animals per group. Measurements of relative liver weight, DNA, protein, respiration, respiratory control, mitochondrial membrane permeability, mixed function oxidases (N-demethylation, aromatic hydroxylation, and cytochrome P-450), glucose-6-phosphatase, diene conjugates, and protein binding were carried out. Mirex caused an increase in relative liver weight, total liver DNA, total protein in each liver fraction, mitochondrial respiration and coupling, and mixed function oxidases. Except for DNA, each parameter increased with an increase in the level of Mirex in the diet and the duration of feeding. DNA was stimulated at all doses to 130–150% of control levels. A further rise in DNA occurred during the onset of the development of liver nodules. The action of Mirex on mitochondria occurred predominantly in the light mitochondrial fraction and resulted in a stimulation of respiration, increased coupling and a decrease in membrane permeability. Mirex added to control mitochondria (in vitro) produced none of these changes. Glucose-6-phosphatase activity was decreased with increasing levels of Mirex in the diet and duration of feeding. The concentration of microsomal diene conjugates was not affected by Mirex. Mirex was formly bound to soluble liver proteins both in vivo and in vitro, but did not displace cortisol from the cortisol receptor protein.

Reversible inhibition of spermatogenesis in rats and monkeys with a new class of indazol-carboxylic acids.

Abstract The effect of oral administration of AF 1312 TS upon the testicular germinal epithelium was studied in the rat and monkey. A single oral dose of 100 or 200 mgm/kgm given to mature male rats was not effective, but five consecutive doses of 200 mgm/kgm produced marked decrease in testicular weight and complete inhibition of spermatogenesis, while the weight and histology of the prostate and seminal vesicles were not affected. Daily doses of 10 mgm/kgm for 37 weeks or five consecutive doses of 50 mgm/kgm for 1 week were ineffective in the monkey. However, when the five dose regimen was followed by single weekly doses of 50 mgm/kgm for 6 months, complete inhibition was achieved and maintained in the monkey after 8 weeks. Daily doses of 100 mgm/kgm for 6 months resulted in inhibition of spermatogenesis. Preliminary studies with AF 1890 (an analog of AF 1312 TS ) given at levels of 50 mgm/kgm for 5 days to rats resulted in complete inhibition of spermatogenesis. The activity of this analog was four times greater than AF 1312 TS . In monkeys, a daily dose of 200 mgm/kgm for 2 weeks also resulted in suppression of spermatogenesis.

Carrageenan: the effect of molecular weight and polymer type on its uptake, excretion and degradation in animals.

Abstract A variety of τ-, κ- and λ-carrageenans was given to guinea-pigs, monkeys and rats, either in the drinking-water, by gavage or in the diet. Faecal and liver samples were examined qualitatively by gel electrophoresis, to determine any changes in the apparent molecular weight of carrageenans after administration. Quantitative measurements of carrageenans were carried out on samples of liver and urine. That there was little or no absorption of carrageenans of high molecular weight was evidenced by the absence of carrageenan from the livers of guinea-pigs or rats or from the urine of guinea-pigs or monkeys. By contrast, substantial amounts of carrageenan were found in the livers of guinea-pigs and rats given low-molecular-weight carrageenans (Mn ⩽ 40,000). Intermediate amounts of carrageenan were found in livers of animals given carrageenans ranging in Mn between 40,000 and 150,000. Urinary excretion of carrageenan was limited to low-molecular-weight material (Mn ⩽ 20.000). Qualitative and quantitative evidence indicated that there was an upper limit to the size of carrageenan molecules absorbed, but estimates of this upper limit ranged from 10,000 to 85.000 depending upon the analytical approach. Absorption of carrageenan from the drinking-water may differ qualitatively from absorption from the diet. Analysis of faecal samples by gel electrophoresis showed that degradation of high-molecular-weight carrageenan had occurred, either in the gut or in the faeces.

Environmental chemical-induced immune dysfunction.

Antibody formation, endotoxin sensitivity, and resistance to a challenge malarial infection were evaluated in mice fed a diet containing polychlorinated biphenyl (PCB) (Aroclor 1242) or hexachlorobenzene (HCB). Antibody synthesis to the antigen sheep RBC (SRBC) was significantly depressed in the PCB- and HCB-treated (167 ppm) animals as evidenced by the fact that control mice elicited an approximate twofold increase in antibody formation over the chemical-treated mice. Serum IgA concentrations in the PCB- and HCB-treated mice were consistently 40--80 mg/dl lower than control values. Gram-negative endotoxin (Salmonella typhosa) sensitivity in PCB- and HCB-treated mice was increased 5.2- and 32-fold, respectively, following the dietary administration of 167 ppm of Aroclor 1242 or HCB for 6 weeks. An endotoxin hypersusceptibility was also noted at 3 weeks after dietary administration. Decreased resistance to a malaria challenge was also demonstrated in the xenobiotic-treated mice. A 20% decrease in mean survival time of mice fed Aroclor 1242 for 3 to 6 weeks and inoculated with Plasmodium berghei (NYU-2) was observed. Infected mice which had received HCB for 3 or 6 weeks manifested reductions in mean survival time of 24 and 31%, respectively. The data indicated that environmental chemical contaminants impair host resistance and, since no concomitant histopathological alterations were observed in the treated mice, the evaluation of immune parameters may possibly be a sensitive indicator of toxicity.

Uptake and storage of degraded carrageenan in lysosomes of reticuloendothelial cells of the rhesus monkey, Macaca mulatta☆

Abstract Rhesus monkeys ( M. mulatta ) were given solutions of 1% HMR or of degraded carrageenan (2, 1, or 0.5% C16) as drinking water over a period of 7–11 weeks, followed by recovery for up to 24 weeks. There was no evidence of storage of HMR, in contrast to C16 which produced extensive reticuloendothelial retention of material that was still present in Kupffer cells 6 months after administration of C16 was stopped. The stored material was PAS positive and metachromatic; it occupied sites corresponding to the presence of acid phosphatase and β-glucuronidase. Ultrastructurally, the hepatocytes appeared normal in all groups. In C16-treated monkeys fibrillar material was seen in membrane-bound vacuoles within reticuloendothelial cells, still present after the recovery period. Acid phosphatase reaction product could be demonstrated in these storage vacuoles. While injected horseradish peroxidase was readily taken up in small vesicles and lysosomes of Kupffer cells in control and HMR-treated monkeys, it was excluded from storage vacuoles produced by C16 administration and appeared only in micropinocytic vesicles. The implications of these findings are discussed in terms of the pathological effects of degraded carrageenan and the influence of stored sulfated polysaccharide on lysosomal function.