Absence of toxic effects in F344/N rats and B6C3F1 mice following subchronic administration of chromium picolinate monohydrate.

Chromium picolinate monohydrate (CPM) is a synthetic compound heavily marketed to consumers in the United States for use as a dietary supplement for muscle building and weight loss. The National Toxicology Program (NTP) tested the toxicity of this compound based on the potential for widespread consumer exposure and lack of information about its toxicity. Groups of 10 male and 10 female F344/N rats and B6C3F(1) mice were exposed to 0, 80, 240, 2000, 10,000, or 50,000 ppm CPM in feed for 13 weeks. CPM administration produced no effect on body weight gain or survival of rats or mice. Organ weights and organ/body weight ratios in exposed animals were generally unaffected by CPM. No compound-related changes in hematology and clinical chemistry parameters were observed. There were no histopathological lesions attributed to CPM in rats or mice.

Toxicity of lactide in dogs after 2 and 13 weeks of daily oral dosing.

Two-week and 13-week studies were conducted to determine the toxicity of lactide when the compound is administered orally in gelatin capsules to beagle dogs. In the 2-week study, daily doses of 0, 10, 100, 400, 1000 and 2500 mg/kg body weight/day were administered to dogs of both sexes for 14 consecutive days. All dogs survived to the end of the study. Clinical signs consistent with irritation of the alimentary tract occurred in dogs in the 1000 and 2500 mg/kg dose groups. Reductions in body weight gain and in absolute and relative thymus weights were observed in the same two dose groups, and reductions in absolute and relative spleen weights were seen in the 2500 mg/kg dose group. These changes were considered to be secondary to the stress resulting from irritation of the gastrointestinal tract. Gross and microscopic lesions were indicative of irritation, and included dark foci and haemorrhage of the stomach lining, and erosion and ulceration of the stomach and the oesophagus. Also noted in all high-dose dogs was renal tubular regeneration, which may represent repair of previous necrosis of the tubular epithelium. In the 13-week study, no deaths occurred when dogs were given daily oral doses of 0, 4, 20 or 100 mg/kg body weight/day. No clinical signs of toxicity were observed, and the compound had no effect on body weights, food consumption, or any of the clinical chemistry, haematology or urinalysis parameters assessed. Gross and microscopic findings considered to be potentially related to lactide administration were minimal, and included a stomach focus in one dog of each sex in the 100 mg/kg group. The stomach focus in the 100 mg/kg female dog was manifested microscopically as a stomach ulcer. Based on these results, the primary toxic effect of lactide was considered to be irritation of the gastrointestinal tract, and the no-observed-adverse-effect level (NOAEL) after subchronic oral dosing in dogs was considered to be 100 mg/kg/day.

Toxicity of 3,3',4,4'-tetrachloroazoxybenzene in rats and mice.

The toxicity of 3,3',4,4'-tetrachloroazoxybenzene (TCAOB) was evaluated in 13-week gavage studies in male and female F344/N rats and B6C3F1 mice. In addition to histopathology, evaluations included clinical chemistry, hematology, thyroid hormone analyses, and effects on sperm morphology and estrous cycle length. Groups of 10 rats and 10 mice of each sex were exposed to TCAOB at dose levels of 0, 0.1, 1, 3, 10, or 30 mg/kg 5 days a week for 13 weeks. In the rat studies, the major effects included death in the 30 mg TCAOB/kg dose group; at lower exposure levels, a decrease in body weight gain, a decrease in thymus weight, an increase in liver weight, an increase in hematopoietic cell proliferation in the spleen and liver, a responsive anemia, a decrease in platelet counts, a chronic active inflammation of the vasculature in the lung, an increase in cardiomyopathy, hyperplasia of the forestomach, and a marked decrease in circulating thyroxine concentrations were observed. In male rats a decrease in sperm motility in the epididymides was observed. In addition, in female rats an increase in lung, spleen, kidney, and heart weights and nephropathy was observed. Furthermore, the estrous cycle length was increased. In the mouse studies, the major effects for males and females included a decrease in thymus weights, an increase in liver and kidney weights, centrilobular hypertrophy in the liver, hematopoietic cell proliferation, hyperplasia of the forestomach, and dilatation of hair follicles. The spectrum of effects in both rats and mice after exposure to TCAOB indicates that dioxin-like effects occur in addition to effects that have not been observed with dioxin-like compounds. No no-observed-adverse-effect level was reached in male or female rats or mice.

Toxicity of 3,3',4,4'-tetrachloroazobenzene in rats and mice.

The toxicity of 3,3',4,4'-tetrachloroazobenzene (TCAB) was evaluated in 13-week gavage studies in male and female F344/N rats and B6C3F1 mice. In addition to histopathology, evaluations included clinical chemistry, hematology, thyroid hormone analyses, and reproductive parameters. Groups of 10 rats and 10 mice of each sex were exposed to TCAB at dose levels of 0, 0.1, 1, 3, 10, or 30 mg/kg for 5 days a week for 13 weeks. In the rat studies, the major effects for both males and females included a 10% decrease in terminal body weight at 30 mg/kg/day, an increase in hematopoietic cell proliferation in the spleen at 10 and 30 mg/kg/day, and a responsive anemia at 10 and 30 mg/kg/day. A 15 to 30% decrease in platelet counts and a 20 to 40% decrease in thymus weights was observed at 10 and 30 mg/kg/day. An increase in liver weight up to 15% was found at 3 mg/kg/day and higher doses in males and at 10 and 30 mg/kg/day in females, respectively. An increase in spleen weights up to 15% was observed at 10 and 30 mg/kg/day in males and at 30 mg/kg/day in females. A marked decrease in circulating total thyroxine (TT4) was found in both males and females at all dose levels tested. TT4 could hardly be detected at 10 and 30 mg TCAB/kg/day. In addition, hyperplasia of the forestomach was increased at 3 mg/kg/day and higher doses in males and at 30 mg/kg/day in females. In the mouse studies, an increase in liver and spleen weight was observed up to approximately 25% in both males and females at 10 and 30 mg/kg/day. Hyperplasia of the forestomach was observed at 1 mg/kg/day and higher doses in both males and females. In males, a 30% decrease in thymus weights at 30 mg/kg/day and a 60% decrease in epididymal sperm density at 3 and 30 mg/kg/day was observed. Also in males, centrilobular hypertrophy of hepatocytes and an increase in hematopoietic cell proliferation in the spleen was observed at 3 mg/kg/day and higher doses. Based on the current study and information in the literature, TCAB has dioxin-like properties. Comparison of the effects of TCAB in the present study and in the literature to those with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) indicates that TCAB is from two to six orders of magnitude less potent than TCDD depending on the end point.

Comparison of the toxicity of cinnamaldehyde when administered by microencapsulation in feed or by corn oil gavage.

The toxicity of cinnamaldehyde (CNMA) was compared after administration by gavage and in dosed feed. Rats and mice of both sexes received CNMA by daily corn oil gavage (for 2 wk), or in microencapsulated form in feed (2 wk for rats, 3 wk for mice). Feed formulations contained 0-10% CNMA microcapsules, equivalent to approximate daily doses of 0-3000 mg CNMA/kg body weight for rats and 0-10,000 mg CNMA/kg body weight for mice. Concentrations were chosen to deliver CNMA doses approximately equal to doses in the gavage study. Gavage doses of 2620 mg/kg/day and above in mice and 940 mg/kg/day and above in rats produced nearly 100% mortality; there were no deaths in animals receiving microencapsulated CNMA. Rats and mice receiving CNMA in feed showed a dose-related decrease in body weight gain, which was accompanied in rats by hypoplastic changes in reproductive organs and accessory sex glands. CNMA administration by either route caused hyperplasia of the forestomach mucosa. These results demonstrate that microencapsulation in feed can present a useful alternative to gavage dosing for repeated-dose or prolonged-exposure studies, in that (1) the toxic effects of CNMA were similar after gavage dosing and after administration in microencapsulated form in feed, (2) ingestion of chemical in the feed more closely approximates human exposures, and (3) microencapsulation allows the delivery of higher net doses of chemical, while avoiding the acutely toxic effects of a bolus dose.

Subchronic toxicity of cupric sulfate administered in drinking water and feed to rats and mice.

The effects of acute poisoning by cupric sulfate in a number of species are well known; however, the effects of chronic low-level ingestion of cupric sulfate are less well characterized. Because exposure of humans to cupric sulfate may occur through drinking water, food, soil, or ambient air, subchronic toxicity studies were conducted in male and female F344/N rats and B6C3F1 mice by the drinking water (2-week exposure) and dosed feed (2- and 13-week exposure) routes. Animals were evaluated for histopathology, clinical pathology, reproductive toxicity, and tissue metal accumulation, and target organs were examined by a variety of special stains and by electron microscopy to characterize the observed lesions. In drinking water, cupric sulfate concentrations of 300 to 100 ppm produced no ill effects, whereas concentrations of 3000 to 30,000 ppm were lethal to rats and mice within 2 weeks. In feed, cupric sulfate concentrations of 4000 to 16,000 ppm caused significant reductions in body weight gain in both species in the 2- and 13-week studies. Hyperplasia and hyperkeratosis of the limiting ridge of the forestomach were present in both species in the 2- and 13-week studies. Rats in the dosed feed studies had a dose-related increase in inflammation in the liver and changes in clinical chemistry parameters which were indicative of hepatocellular damage and cholestasis. Histologic changes in the kidneys of rats consisted of a dose-related increase in the number and size of eosinophilic protein droplets in the epithelial cytoplasm and the lumina of the proximal convoluted tubules. Droplets were larger and more numerous in males than in females. Urinalysis results were suggestive of renal tubular epithelial damage. Iron staining of spleens from treated animals indicated a marked depletion of iron stores in both male and female rats, but not in mice, while hematologic and clinical chemistry alterations in rats in the 13-week study, along with histologic changes in bone in the 2-week dosed feed study, were indicative of a microcytic anemia. Cupric sulfate produced no adverse effects on any of the reproductive parameters measured in rats or mice of either sex. These results indicate that cupric sulfate at high exposure levels is a hepatic and renal toxicant, as well as an inducer of anemia in rodents, with rats more sensitive than mice following subchronic exposure.

Inhalation toxicity of 1,6-hexanediamine dihydrochloride in F344/N rats and B6C3F1 mice.

1,6-Hexanediamine (HDA) is a high production volume chemical which is used as an intermediate in the synthesis of paints, resins, inks, and textiles and as a corrosion inhibitor in lubricants. Two- and 13-week studies of the toxicity of the dihydrochloride salt of HDA (HDDC) were conducted in male and female Fischer 344/N rats and B6C3F1 mice using whole-body inhalation exposure. Both species were evaluated for histopathologic and reproductive effects, and rats were examined for clinical chemistry and hematologic changes. In the 2-week inhalation studies, animals were exposed to 10-800 mg HDDC/m3, 6 hr per day. All rats, all female mice, and two of five male mice in the high-exposure group died before the end of the study. Surviving mice in this group had a dose-dependent depression in body weight gain. Clinical signs were primarily related to upper respiratory tract irritation and included dyspnea and nasal discharge in both species. Treatment-related histopathologic lesions included inflammation and necrosis of the laryngeal epithelium of both species and the tracheal epithelium of mice, as well as focal inflammation and ulceration of the respiratory and olfactory nasal mucosa. In the 13-week inhalation studies, animals were exposed to HDDC at concentrations of 1.6-160 mg/m3 for 6 hr per day, 5 days per week. In addition to the base study groups, a supplemental group of rats at each exposure level was included to assess the effect of HDDC on reproduction. No treatment-related changes in organ weights or organ-to-body-weight ratios occurred in rats, and no treatment-related clinical signs or gross lesions were seen in either species. Chemical-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) in the two highest exposure groups and were similar in both species. These lesions included minimal to mild focal erosion, ulceration, inflammation, and hyperplasia of the laryngeal epithelium, in addition to degeneration of the olfactory and respiratory nasal epithelium. HDDC caused no significant changes in sperm morphology or vaginal cytology and no significant adverse effects on reproduction in rats or mice. Hematologic and clinical chemistry changes in rats were minor and sporadic and were not accompanied by related histologic findings. HDDC did not increase the frequency of micronucleated erythrocytes in mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of murine cell lines from diethylstilbestrol-induced uterine endometrial adenocarcinomas.

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice. Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormone-dependent lesions in 18-mo.-old mice. Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear:cytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli. After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen. Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes. Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three- to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line. These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.

Role of transforming growth factor beta in the proliferative effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on human squamous carcinoma cells.

The highly toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has recently been shown to stimulate proliferation of two human squamous carcinoma cell lines, SCC-15G and SCC-25, by decreasing the sensitivity of the cells to high density growth arrest. TCDD is known to alter the activity of several endogenous growth-regulatory compounds, and this study was undertaken to investigate the possibility that modulation of transforming growth factor beta (TGF-beta) activity might be involved in the mechanism of growth stimulation by TCDD. TGF-beta inhibited monolayer growth and DNA synthesis of SCC-15G and SCC-25 cells equally well in the presence and the absence of 10 nM TCDD. TCDD alone stimulated proliferation and inhibited differentiation in both cell lines but had no effect on binding of 125I-TGF-beta to or secretion of TGF-beta by SCC-15G cells. Inhibition of growth of SCC-15G cultures by TGF-beta was incomplete, in that cell number continued to increase even in the presence of 100 pM TGF-beta, although at a greatly reduced rate compared to non-TGF-beta-treated controls. These cells, grown in 100 pM TGF-beta alone, reached growth arrest at the same density as non-TGF-beta-treated cultures but failed to reach density-dependent growth arrest when treated with TCDD. Cells treated for 12 h with TCDD exhibited maximal induction of ethoxyresorufin-O-deethylase activity. TGF-beta inhibited this induction in a dose-dependent manner even when added after a 12-h TCDD pretreatment. The inhibition was rapidly reversible after removal of TGF-beta, indicating that it occurred via a mechanism which did not involve inhibition of very early steps in the TCDD response pathway. These results demonstrate that the stimulatory effect of TCDD on keratinocyte proliferation is not mediated through alterations in TGF-beta activity and that TCDD and TGF-beta appear to exert their opposite effects on cellular proliferation by independent mechanisms.

Inhibition of high-density growth arrest in human squamous carcinoma cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The highly toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent carcinogen and tumor promoter, and affects cellular proliferation and differentiation both in vivo and in vitro. This report presents data showing that TCDD enhances the proliferation of two human squamous carcinoma cell lines in monolayer culture by inhibiting growth arrest at high cell density. SCC-15G and SCC-25 cells were treated with 0-100 nM TCDD in culture medium, and examined for changes in proliferation and differentiation. TCDD stimulated increases in cell number and DNA synthesis of both cell lines, and inhibited differentiation of SCC-15G cells in a dose-dependent manner. The minimum effective concentrations for increases in proliferation were 0.1 nM in SCC-15G cells and 1 nM in SCC-25G cells. The saturation density of SCC-15G cells grown in 10 nM TCDD was approximately double that of untreated controls, while the saturation density of SCC-25 cells was 50% above controls. TCDD-induced increases in proliferation were detectable only in cells exposed at subconfluent density, then assayed after control cultures had reached high-density growth arrest. There was no difference in cell number or DNA synthesis between control and TCDD-treated cultures when cells were both treated and assayed during the logarithmic phase of growth, nor in cultures treated after the cells had reached high density growth arrest. Therefore, TCDD-induced proliferation resulted from failure of treated cells to undergo normal density-dependent growth arrest rather than from direct mitogenic stimulation of the cells. Differentiation (envelope competence and keratin staining) of SCC-15G cells was inhibited by TCDD, despite the fact that in these cultures cell density was twice that of the controls. The sensitivity of SCC-15G cells to modulation of growth and differentiation by TCDD provides the basis for a model to examine the biological mechanisms of TCDD-induced alterations in proliferation and differentiation of epidermal cells.

Relative toxicity and tumor-promoting ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PCDF), and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) in hairless mice.

2,3,7,8-Tetrachlorodibenzo-p-dixoin 2,3,4,7,8-pentachlorodibenzofuran (PCDF), and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) are highly toxic members of a class of environmental contaminants, the polychlorinated aromatic hydrocarbons (PCAH), which exhibit a similar and highly characteristic spectrum of toxic effects. For purposes of risk assessment, it is important to be able to make accurate estimates of the relative potency of these and related compounds. Previous investigations have indicated that, in acute exposure or in vitro studies, PCDF is approximately 0.1 times as toxic and HCDF is approximately 0.01 times as toxic as TCDD. In this study, we compared the relative toxicity and tumor-promoting abilities of TCDD, PCDF, and HCDF in hairless mouse skin. Female hairless mice (HRS/J hr/hr) were treated dermally with the initiator MNNG, then dosed twice weekly for 20 weeks with acetone, TCDD (2.5-10 ng/mouse/dose), PCDF (25-100 ng/mouse/dose), or HCDF (250-1000 ng/mouse/dose) as promoter. TCDD, PCDF, and HCDF were all potent promoters for the induction of squamous cell papillomas. There was, however, no difference in the incidence or multiplicity of papilloma formation between groups. The same doses of the three PCAH, in the absence of initiator, induced no skin papillomas. TCDD produced a significant increase in liver:body weight ratio (p less than 0.001) at all doses and a decrease in thymus:body weight ratio at a dose of 10 ng (p less than 0.001). Mice treated with PCDF and HCDF had marked thymic and splenic involution, liver hypertrophy, mucous cell hyperplasia in the fundic portion of the glandular stomach, and loss of body weight. PCDF and HCDF produced a greater incidence and severity of dermatotoxic effects than TCDD. Based on data for dermal toxicity and changes in body weight and organ weights, PCDF is estimated to be 0.2 to 0.4 times, and HCDF 0.08 to 0.16 times, as toxic as TCDD following repeated dermal exposure. Therefore, toxic equivalence factors generated using data from acute and/or in vitro studies may underestimate the risk from repeated low-dose exposures to these compounds.

Lack of correlation between sensitivity to growth inhibition and receptor number for transforming growth factor beta in human squamous carcinoma cell lines.

Four human squamous carcinoma cell lines, SCC-9, SCC-12F, SCC-15G, and SCC-25, were examined for sensitivity to the growth-inhibitory and differentiation-inducing effects of transforming growth factor beta (TGF-beta). None of the four cell lines was induced to differentiate, as measured by staining for keratin and by competence of cells to form cross-linked envelopes, by concentrations of TGF-beta as high as 1000 pM. TGF-beta did not inhibit DNA synthesis or proliferation of SCC-12F cells in monolayer culture. Monolayer growth and DNA synthesis of SCC-15G and SCC-25 cells were markedly inhibited by 10 pM TGF-beta, with maximal inhibition between 10 and 100 pM. Inhibition of SCC-15G cells was apparent as early as 8 h after addition of TGF-beta, but inhibition of SCC-25 cells was not measurable until 2 days. Growth inhibition of SCC-15G cells was completely reversible, whereas inhibition of SCC-25 cells was irreversible. When growth of cells was measured in a defined medium supplemented with 0.5% fetal bovine serum, TGF-beta inhibited both the monolayer and clonal growth of SCC-15G cells. The monolayer growth, but not the clonal growth, of SCC-25 cells was inhibited. Growth of SCC-9 cells in confluent cultures was slightly inhibited by TGF-beta, while growth in subconfluent cultures was unaffected. All four cell lines, when assayed for binding of 125I-labeled TGF-beta, displayed high-affinity (KD = 2-44 pM) binding sites. These sites were present in very low numbers (less than 2000 sites/cell) in SCC-9, SCC-15G, and SCC-25 cells. SCC-12F cells contained 2000-5500 high-affinity sites/cell. Thus, a lack of sensitivity to growth inhibition by TGF-beta does not necessarily correlate with an inability to bind the growth factor to specific cell surface receptors. The possibility remains that altered sensitivity to TGF-beta may be due to loss of or changes in the relative proportions of the various TGF-beta receptor types. TGF-beta is believed to play a primary role in control of normal cellular growth and differentiation, and the study of cells with defective responses to this growth factor should help to identify some of the critical points within the growth cycle at which malignant cells may escape normal regulatory controls. The differential responses of these four human squamous carcinoma cell lines to TGF-beta should provide a useful model for studying such control mechanisms.

The influence of aging on intestinal absorption of TCDD in rats.

The effects of age on intestinal absorption of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied using adult male Fischer-344 rats of 3 different age groups: 13 weeks old (young), 13 months old (mature), and 26 months old (senescent). Absorption was measured with an in situ intestinal recirculation perfusion procedure. Absorption expressed in terms of ng TCDD absorbed/g intestinal dry weight/h was 166, 149 and 143 ng/g/h in the young, mature and senescent groups, respectively. When absorption was calculated in terms of ng TCDD absorbed/g mucosal dry weight/h, the decrease between the senescent rats and the 2 younger age groups, from 544 ng/g/h (young) to 351 ng/g/h (senescent), was not statistically significant (P less than 0.05). It was demonstrated that absorption of TCDD was unaffected by the presence of 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) in the perfusate, but that HCB absorption was (P less than 0.01) enhanced by the presence of TCDD.

Determination of the oral toxicity of Sesbania drummondii seeds in chickens.

Extracts of Sesbania drummondii administered to chickens by oral intubation are lethal within several days. Effects are dose-dependent; a dose of 1% of body weight is uniformly lethal in 5 days. Signs of poisoning include weakness, depression (CNS), anorexia, diarrhea, ruffled feathers, cold feet, and rapid loss of body weight. Microscopic examination indicates damage to kidney glomeruli and leakage of protein into the kidney tubules. Packed cell volume and plasma glucose concentrations show no difference between controls and treated chickens; however, creatine kinase is increased and plasma cholinesterase and total plasma protein values are severely depressed in poisoned birds. Neither a specific toxin nor a mechanism of action for toxicity has yet been identified.

Assessment of smooth muscle function in Sesbania drummondii toxicosis in Gallus domesticus.

An in vitro assessment based on tissue responsiveness to 2 agonists-histamine and carbachol-was made on smooth muscle activity in chickens experimentally poisoned with sesbania. Crude extracts of Sesbania drummondii were prepared and 2 dosage levels, 0.25% and 0.5% of body weight, were used. The birds were dosed orally with the extract for 3 consecutive days, and on the 4th day segments of ileum and lung were collected from each bird. The isometric contractions of each tissue, produced by the addition of histamine or carbachol in graded concentrations, were recorded. The cumulative concentration-effect curves for the tissues to the agonists were constructed and compared with respective control curves. The results indicated the responsiveness of the tissues in the treated groups was significantly decreased, compared with that of tissues in the controls. Responses of both intestinal and parenchymal strips in the chickens given the higher dosage (0.5%) were decreased significantly, whereas in those given the smaller dosage (0.25%), only parenchyma had a significant response. This indicates that the activity of smooth muscles in general was depressed by sesbania. The effect was more evident in the lung than in the intestine. Therefore, an active principle in the extract which affects smooth muscle, rather than causing direct irritation, may exist. This assessment of smooth muscle activity is sensitive and was effective in detecting changes in tissues from sesbania-treated birds which had not shown any clinical signs. The results also support the possibility that smooth muscle involvement could be a primary cause of toxicity in sesbania poisoning.

Effects of a Cassia occidentalis extract in the domestic chicken (Gallus domesticus).

The effects of an orally administered Cassia occidentalis extract were studied in chickens. A 25 mM sodium bicarbonate solution effectively extracted the toxic principle. Toxic activity was reduced, but not eliminated, when the heated extract (90 C, 40 minutes) was administered. The toxic principle was in the pellet after centrifuging the extract at 38,000 X g. Daily administration of the extract produced weight loss and muscular weakness. Microscopic examination revealed skeletal and cardiac muscle degeneration and hepatocyte vacuolation. Electron microscopic examination revealed mitochondrial disruption. Respiratory studies on liver mitochondria isolated from treated chickens demonstrated lower phosphorylation ratios, lower respiratory control ratios, and lower rates of oxygen use.

Preliminary isolation of a myodegenerative toxic principle from Cassia occidentalis.

Aqueous and organic extractions of ground seeds of Cassia occidentalis were obtained. Chickens were dosed with extracted material to assess the toxicity of the extracts. Organic extracts with methanol, ethanol, chloroform, ethyl acetate, and benzene were ineffective in removing the toxin from the seeds. Aqueous extractions, using 25 mM sodium bicarbonate or 250 mM sodium citrate, removed the toxin from the seeds, but left the toxin bound to particulate matter in the extract. Addition of Triton X-100 to the aqueous buffers effectively solubilized the toxin from the particulate matter. Signs of intoxication in the chickens were loss of weight, weakness, diarrhea, hypothermia, occasionally ataxia, and recumbency; then death. Gross lesions included paleness of skeletal and cardiac muscles and congestion of the liver. Microscopic lesions in muscle tissue were vacuolation, proliferation of sarcolemmal nuclei, and separation of myofibrils. Electron microscopic examination revealed disruption of mitochondrial cristae and swelling and rupture of mitochondria.

Determination of the oral toxicity of Cassia obtusifolia seeds in chickens.

Chickens were fed dried seeds of Cassia obtusifolia, either whole or ground, mixed in with regular chicken ration. Other chickens were dosed at 2% of body weight with either an aqueous extract of seeds, or the pellet or supernatant from an 18,000 X g centrifugation of the extract. Weight gains were only slightly lower in chickens treated with up to 10% seeds in the feed. This was attributed to reduced food intake rather than to toxic effects of the plant. Necropsy examinations of chickens from all groups revealed none of the lesions typical of intoxication with Cassia. Histologic examinations revealed no lesions.