Inhalation two-generation reproductive toxicity study of methyl isobutyl ketone in rats.

To evaluate whether methyl isobutyl ketone (MIBK) affects reproductive performance, a two-generation reproduction study was conducted. MIBK was administered to 30 Sprague-Dawley rats/sex/group via whole-body inhalation at concentrations of 0, 500, 1000, or 2000 ppm, 6 h daily, for 70 days prior to mating. F(0) and F(1) females were exposed from mating through gestation day 20 and from postnatal day 5; F(2) litters were maintained through postnatal day 21. No treatment-related mortality of adult animals occurred. There was a dose-related increase in adult animals with no or a decreased response to a sound stimulus at 1000 and 2000 ppm; however, no adverse clinical signs occurred 1 h after exposure, suggesting this was a transient sedative effect. Clinical signs of central nervous system (CNS) depression in the pups were observed and one F(1) pup died after initial exposure to 2000 ppm on postnatal day 22; subsequently exposure was delayed until postnatal day 28. Decreased body weight gain and slight decreased food consumption were observed during the first 2 weeks of exposure in both generations at 2000 ppm. There were no adverse effects on male and female reproductive function or landmarks of sexual maturation. Increased F(0) and F(1) liver weights with associated centrilobular hypertrophy occurred in rats at 2000 ppm, indicative of an adaptive response. Increased male kidney weights at all exposure concentrations, associated with hyaline droplets, were indicative of male rat-specific nephropathy. Other than acute sedative effects, the no-observed-adverse-effect level (NOAEL) for parental systemic effects (excluding male rat kidney) was 1000 ppm, based on transient decreased body weight and food consumption; for reproductive effects, 2000 ppm, the highest concentration tested; and for neonatal toxicity, 1000 ppm (based on acute CNS depressive effects).

The effect of repeated methyl iso-butyl ketone vapor exposure on schedule-controlled operant behavior in rats.

Methyl iso-butyl ketone (MiBK), a commercial solvent, was selected by the US Environmental Protection Agency (US EPA) for testing under the Multi-Substance Rule for the Testing of Neurotoxicity (US EPA, 1993) using schedule-controlled operant behavior (SCOB) to determine if subchronic exposure to MiBK vapor had the potential to alter behavior as an indicator of neurotoxicity. Food-restricted and ad libitum-fed Sprague-Dawley male rats were exposed to 0, 250, 750, or 1500 ppm MiBK for 6 h/day, 5 d/wk for 13 weeks. SCOB testing of food-restricted animals, using a multiple fixed ratio (FR)/fixed interval (FI) schedule (FR20:FI120), was conducted prior to each exposure to maintain the operant behavior; the data from Weeks -1, 4, 8, and 13 were evaluated for evidence of neurotoxicity. SCOB testing was also evaluated for two weeks following the cessation of exposures. Ad libitum-fed animals were included to assess systemic effects using routine indicators such as changes in body weight, food consumption, and organ weight. No significant differences were seen in fixed-ratio run rate, FR pause duration, fixed-interval response rate, and index of curvature values at any concentration. Animals exposed to 750 and 1500 ppm MiBK exhibited clinical signs associated with transient reduced activity levels, but only during exposure. No signs of reduced activity were observed immediately after exposure for either group. No other treatment-related abnormalities were observed during exposure. Food-restricted animals did not demonstrate any increased or decreased sensitivity to the CNS depressive effects of MiBK relative to the ad libitum-fed animals. No treatment-related body weight differences were observed within either the food-restricted groups or the ad libitum-fed groups, although body weights of the former were clearly depressed compared with those of the latter. Relative and absolute liver, and relative kidney weights were significantly greater for the 750 and 1500 ppm ad libitum-fed animals. No differences in kidney weight were observed for food-restricted animals, but absolute and/or relative liver weights were significantly higher for all the treated food-restricted groups. The results of this study indicate that repetitive exposures to high concentrations of MiBK vapors do not result in adverse effects on operant behavior in the rat.

2-Ethylhexanoic acid: subchronic oral toxicity studies in the rat and mouse.

Groups of 10 male and 10 female Fischer 344 rats and B6C3F1 mice were fed diets containing either 0.0, 0.1, 0.5 or 1.5% 2-ethylhexanoic acid (EHA) for 13 wk. Additional groups of 10 male and 10 female rats or mice. were fed either 0.0 or 1.5% EHA for 13 wk followed by a 4-wk recovery (non-treatment) period. Based on food consumption and body weight, the EHA diets provided doses of 61, 303 or 917 mg/kg/day for male rats and 71, 360 or 1068 mg/kg/day for female rats. The EHA diets provided doses of 180, 885 or 2728 mg/kg/day for male mice and 205, 1038 or 3139 mg/kg/day for female mice. No mortality or significant clinical signs of toxicity were observed during the study. Body weights and food consumption of both rats and mice fed 1.5% EHA were lower beginning after the first week of treatment, consistent with a reduction in food consumption. Other groups were unaffected by treatment. After 13 wk, lower triglyceride levels occurred in male mice fed 1.5% EHA and female mice fed 0.5 or 1.5% EHA, but not in other groups. Cholesterol levels were higher in all male rat test groups and in female rats and male and female mice fed either 0.5 or 1.5% EHA, although this effect was reversible following a 28-day recovery period. The principal effects of EHA involved the liver or metabolic processes associated with the liver. The 0.5 and 1.5% diets in both rats and mice were associated with increased relative liver weight and histological changes in hepatocytes, specifically hepatocyte hypertrophy and reduced cytoplasmic vacuolization. Observed histopathological and clinical pathological changes were reversible following recovery. These results indicate that EHA does not produce persistent. overt toxicity in rats or mice following subchronic dietary exposure at concentrations up to 1.5% in feed. The no-observed-adverse-effect level (NOAEL) for male rats was 61 mg/kg/day and the no-observed-effect level (NOEL) for female rats was 71 mg/kg/day, while 180 and 205 mg/kg/day represent NOELs for male and female mice, respectively.

Subchronic 90-day oral toxicology of di(2-ethylhexyl) terephthalate in the rat.

Di(2-ethylhexyl) terephthalate (DEHT), the 2-ethylhexyl diester of terephthalic acid (1,4-benzenedicarboxylic acid) was administered in the diet to groups of 20 male and female Sprague-Dawley rats for 90 days at 1.0, 0.5, 0.1 or 0.0% by weight. No major organ or systemic toxicity resulted from consumption of the diets in any group of animals. Changes that were observed included slight effects on some haematology parameters including haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin at the 1.0% dose; and slight increases in relative liver weights (11.2% in the males, 8.9% in the females), also at the 1.0% dose level. Marginal changes of less than 3% in some red blood cell indices were observed at the 0.5% dietary dose level; however no anaemia or changes in relative liver weights were observed at this dose level. Thus, no significant adverse effects attributable to the test material were identified in animals consuming the two lower doses. In a morphometric study of liver sections, DEHT was found not to induce hepatic peroxisomes at the 1.0% dose level. The positive control material (2-ethylhexanol at 1000 mg/kg, 5 days/wk for 3 wk), produced a 30% increase in liver-to-body weight ratio, and increases in peroxisome cell fraction, and in peroxisome density. The no-effect levels of DEHT in rats consuming the material for 90 days in the diet were 277 and 309 mg/kg/day in males and females, respectively. These results are contrasted with those reported for di(2-ethylhexyl) phthalate (DEHP) in similar feeding studies. While DEHP at 1% in the diet is reported to produce significant effects on the liver, testes, kidney, brain, stomach and adrenal weights, DEHT has been shown in this study to have only a minor effect on liver weight in 90 days at 1.0% in the diet.

Peroxisome induction studies on di(2-ethylhexyl)terephthalate.

Groups of five male and five female rats were fed diets containing from 0% to 2.5% di(2-ethylhexyl)terephthalate (DEHT) or 1.2% di(2-ethylhexyl)phthalate (DEHP) for 21 days. Feed consumption and body weight gains were collected and, at study termination, animals were examined for alterations in body weight, differences in serum lipids, changes in the activities of certain enzymes associated with fat metabolism, and proliferation of hepatic peroxisomes. Feed consumption and weight gain were greatly decreased in DEHT-fed animals only at 2.5%. No biologically significant alterations in absolute liver weight occurred with DEHT. Relative liver weights were increased at 2.5% in both sexes and at 1.0% and 1.2% in females. The alterations were due wholly to decreased terminal body weights. Serum triglyceride and cholesterol levels were not found useful in interpreting the effects of DEHT. Cyanide-insensitive palmitoyl CoA oxidation and lauric acid 11- and 12-hydroxylation were increased in animals consuming 2.5%, but no lower levels of DEHT. Induction of hepatic peroxisomes did not occur at 1.2% DEHT. Interpretation of minimal peroxisomal effects with 2.5% DEHT was confounded by reduced feed consumption. Slight decreases in weight gain occurred in males consuming the 1.2% DEHP diet, but differences were minor relative to effects observed at 2.5% DEHT. Results with DEHP contrasted with those obtained with DEHT. Absolute and relative liver weights, activities of enzymes of lipid metabolism, and peroxisome content were all significantly increased at 1.2% DEHP. Reduction of feed intake was implicated in the effects observed at 2.5% DEHT, since the amount of DEHT consumed by 2.5% animals was only 1.4 times as much as by 1.2% animals. A possible explanation for the observed differences between DEHP and DEHT was related to the results of a metabolic fate study on DEHT. Metabolism of DEHT by the rat appears to occur via rapid hydrolysis of both ester linkages to give two moles of 2-ethylhexanol and one mole of terephthalic acid. Although 2-ethylhexanol has been shown to induce peroxisome proliferation, it appears to be less active in this respect than the monoester of DEHP. The relatively smaller amounts of monoester produced during the metabolism of DEHT may explain the differences seen in these experiments.

Tumor promotion studies in rat tracheal epithelium.

The tracheal epithelium of the Fischer 344 rat is histologically very similar to that of the human bronchus. Also, carcinomas of tracheal origin in F-344 rats are similar in morphology to human bronchogenic carcinomas. Tumor promotion in rat tracheal epithelium was studied by using two model systems. The first is a heterotopic transplant system in which rat tracheas are implanted subcutaneously on the backs of isogenic recipents. In the first system, the epithelium was topically exposed to pellets containing 7,12-dimethylbenz(a)anthracene (DMBA), used as the initiating agent, followed by pellets containing the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the promoting agent. After 98 weeks, a three- to fourfold increase in the percentage of tracheas having malignant tumors was seen in tracheal transplants receiving both DMBA and TPA compared to DMBA alone. Exposure of the tracheal grafts to TPA alone resulted in epithelial hyperplasia and inflammation, but no dysplastic lesions. The second system is an organ culture-cell culture system in which small pieces of trachea are grown in organ culture, then epithelial cells are grown from these pieces as primary cell cultures. The organ cultures were exposed to the direct alkylating agent, N-methyl-N'-nitro-N -nitrosoguanidine (MNNG) used as the initiator, then multiple short exposures to TPA were used to promote. Primary cell cultures and cell lines were then established from these explants. After 52 weeks, a five-fold increase in the percentage of explants producing tumorigenic cell lines was observed when MNNG + TPA-exposed explants were compared to MNNG-exposed explants. Tracheal explants exposed to TPA alone produced many cell lines but none tested were tumorigenic. These two systems provide a means to study tumor promotion in respiratory epithelium. The evidence more importantly suggests that airborne promoting substances may play a key role in the development of bronchogenic carcinoma.

Determination of cocarcinogenic activity of benzo[e]pyrene for respiratory tract mucosa.

As benzo[e]pyrene (B[e]P) has been shown to enhance the carcinogenic effects of benzo[a]pyrene (B[a]P) on mouse skin [26], it therefore seemed important to determine if it would also act as a cocarcinogen in another target tissue, namely respiratory tract mucosa. Tracheal mucosa of rats was concomitantly exposed to B[a]P and B[e]P for up to 6 months. At B[a]P/B[e]P ratios of 1 : 1 and 0.5 : 1, B[e]P had no cocarcinogenic effects on tracheal epithelium. At the higher B[a]P/B[e]P ratio, B[e]P appeared to reduce the carcinoma incidence from 65% (B[a]P alone) to 40% (B[a]P plus B[e]P). In sharp contrast to the carcinoma incidence, the tracheal and peritracheal sarcoma incidence was enhanced 2-3-fold by B[e]P. Thus, while not cocarcinogenic for tracheal epithelium, B[e]P was cocarcinogenic for connective tissue. Together with other results [26], these point to the importance of the target tissue as a determining factor of cocarcinogenic activity of test substances.

Studies of neoplastic development in respiratory tract epithelium.

The dynamics of neoplastic development in conducting airways were studied in an animal model using morphologic and tissue culture techniques. Evidence for the regression of many metaplastic-dysplastic lesions, including advanced "preneoplastic" lesions, was provided. This regression of lesions is not synonymous with reversion of the neoplastic process. The number of "carcinogen-altered" cells and the number of cells with neoplastic potential continued to increase as a function of time after carcinogen exposure. With the cell culture methods developed for these studies, it is possible to analyze qualitatively and quantitatively the progression of the neoplastic process as it takes place in vivo and to detect and enumerate the progenitor cells of later-appearing cancers. The investigations also provide strong evidence suggesting that carcinogen-exposed organs contain many more cells with neoplastic potential. This expression may, however, be sharply enhanced when permissive or promoting conditions prevail. The investigations open up new avenues to develop means for detection of preneoplastic cell populations and for therapeutic intervention during early phases of the neoplastic disease process.

Promotion-like enhancement of tracheal carcinogenesis in rats by 12-O-tetradecanoylphorbol-13-acetate.

Experiments were conducted to determine whether two-stage carcinogenesis could be observed in rat tracheal epithelium using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter. Heterotopic tracheal transplants in Fischer 344 rats were first exposed to 188 microgram of DMBA delivered over a four-week period and subsequently to 100 microgram of TPA. The TPA was released from beeswax pellets at a rate of 1.1 microgram/day during the first two months and at a rate of 0.3 microgram/day for the subsequent two months. TPA alone caused marked inflammation and epithelial hyperplasia in tracheal grafts but no metaplastic or dysplastic changes. The tumor incidence in tracheas exposed to DMBA only was 20%; that in tracheas exposed to DMBA followed by TPA was 72%. TPA also accelerated the appearance of tumors. The mean tumor induction time in the group exposed to DMBA only was 91 weeks as compared to 75 weeks in the group exposed to DMBA and TPA. The data indicate that TPA enhances the tracheal tumor response in a manner similar to that of tumor promotion in mouse skin.

Two-stage carcinogenesis studies with asbestos in Fischer 344 rats.

The cocarcinogenic effect of chrysotile asbestos was investigated in heterotopic tracheal transplants of F344 rats. Tracheal transplants were first exposed to graded doses of dimethylbenz[a]anthracene (DMBA) contained in intraluminal pellets. The doses ranged from 12.5 to 100 micrograms. Control tracheas received blank pellets. Four weeks after the start of DMBA exposure (when all carcinogen had been released from the pellets), the spent pellets were removed, and 200 micrograms chrysotile, a nontumorigenic dose of asbestos, was introduced into the lumina of the preexposed tracheas. No significant enhancement of the tumor response was seen with 100 micrograms DMBA, a dose that was tumorigenic by itself. However, with 50 and 25 micrograms DMBA, nontumorigenic dose levels, a 15 and 23% incidence of tracheal carcinomas occurred when DMBA exposure was followed by a nontumorigenic dose of chrysotile. At 12.5 micrograms DMBA, this effect was not observed. Whatever the mechanisms were that formed the basis of this tumor enhancement effect of asbestos, the consequences were similar to those observed with tumor promotion in classical two-stage carcinogenesis studies.

Toxic and tumorigenic effects of asbestos on tracheal mucosa.

The purpose of the studies was to examine the acute, chronic, and neoplastic effects of asbestos on mucosa of conducting airways. Heterotopic tracheal grafts were used as the experimental model. In the system employed, the test substance was introduced into the airway lumen and kept in intimate contact with the respiratory tract mucosa for weeks or months. UICC chrysotile A and crocidolite asbestos caused marked acute changes of the tracheal mucosa: epithelial hyperplasia, hypersecretion of mucus, and submucosal inflammation. With chrysotile, the hypersecretory state and goblet cell hyperplasia persisted for many months, resulting in a marked distension of the tracheal grafts. Other manifestations of chronic irritation were focal squamous metaplasias, epithelial erosions, and connective tissue changes involving the tracheal submucosa and adventitia as well as the host subcutaneous tissues. The connective tissue changes were characterized by formation of granulomas, often with necrotic centers. In a 29-month tumor-induction study carried out with chrysotile A, the total tumor incidence in 40 tracheal grafts was 40 percent; this included 2 squamous cell carcinomas (5%)(, and 14 sarcomas (35%) as compared with no carcinomas and 6 sarcomas (12%) in 49 vehicle-treated controls. Thus our studies show that chrysotile (1) causes chronic irritation of the tracheal mucosa, (2) augments sarcoma development, and (3) is a weakly but definitely carcinogenic agent for the respiratory tract mucosa.

Quantitative analysis of disturbed cell maturation in dysplastic lesions of the respiratory tract epithelium.

Autoradiographic patterns of [3H]thymidine incorporation, nuclear/cytoplasmic ratios (N/C), and the percentage of dark epithelial cells were analyzed in a group of epithelial lesions induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rat tracheal transplants. It was found that similar lesions of different age exhibit the same labeling indices (LIs), therefore the lesions of different age were subsequently pooled in the following groups and studied by high resolution light microscopic autoradiography: squamous metaplasia without or with only mild atypia, squamous metaplasia with moderate atypia, squamous metaplasia with severe atypia, carcinoma in situ, and microinvasive carcinoma. Normal tracheal and esophageal epithelia were also analyzed. Whereas the normal tracheal basal layer exhibited an LI smaller than 1%, a clear difference between the carcinomas (in situ and invasive) on one hand (LI approximately 32%) and all the remaining epithelia on the other hand (LI approximately 18%) was detected. The LIs of the suprabasal cells exhibited a statistically significant difference between the squamous epithelia without atypia (LI approximately 2%) and the group comprising all the atypical lesions (LI approximately 9%). Gradients of increasing N/C (nucleus-cytoplasm ratios) values could be observed as the lesions increased in severity, especially in the middle and surface layers (e.g., in the surface layer regular metaplasia N/C = 0.08, squamous metaplasia with moderate atypia N/C = 0.26, and carcinoma in situ N/C = 0.50). Dark cells were absent in the normal esophageal epithelium, were present in moderate numbers in the basal layer of regular squamous metaplasia (18%), and increased markedly in the atypical epithelial lesions (approximately 50% in the atypical squamous metaplasias and 70% in carcinoma in situ). In the suprabasal layer dark cells increased from 3% in squamous metaplasia with moderate atypia to 28% in metaplasia with severe atypia and 56% in carcinoma in situ. The results confirm in a quantitative fashion that disturbances of cell maturation and cell proliferation are key features of dysplastic lesions induced by chemical carcinogens, and suggest the use of objective parameters for evaluation and classification of preneoplastic alterations.

Ultrastructural characteristics of carcinogen-induced nondysplastic changes in tracheal epithelium.

Nondysplastic hypotrophic and metaplastic epithelial alterations induced by dimethylbenz(a)anthracene in isogenic tracheal transplants were studied by light and electron microscopy 3--24 months after cessation of a 4-week carcinogen exposure. Hypotrophic epithelium observed at all time points was characterized by the presence of nonciliated cells that adopted either cuboidal or squamous shapes, forming simple or bistratified epithelia. Most of these cells, as well as some metaplastic cells, exhibited features of mucin-secreting cells. The metaplastic epithelia showed nonkeratinizing squamous metaplasia, closely related to transitional metaplasia, and keratinizing squamous metaplasia, which presented either an atrophic or an acanthotic epithelium. Although many of these epithelia showed morphologic features of normal stratified epithelia, several nonkeratinizing squamous metaplasias and acanthotic keratinizing squamous metaplasias exhibited some irregularities, probably representing very early atypical ultrastructural features (ie, perinuclear concentration of tonofilament bundles, the presence of dark and clear basal epithelial cells, interruptions and alterations of the basal lamina). These features were not observed in a group of early squamous metaplasias studied for comparative purposes 2 weeks after cessation of dimethylbenz(a)anthracene exposure, which were characterized by a combination of degenerative phenomena and increased cell proliferation.

Ultrastructural characteristics of carcinogen-induced dysplastic changes in tracheal epithelium.

Squamous metaplasias with various degrees of atypia, carcinomas in situ, and microinvasive carcinomas produced in rat tracheal transplants by a 4-week exposure to demethylbenz(a)anthracene were studied 12-96 weeks after cessation of carcinogen exposure. In addition to the classical nuclear and cytoplasmic indicators of atypism, the squamous metaplasias with slight and moderate atypia were characterized by the presence of glycogen in the spinous layer, atypical composite keratohyalin granules in the granular layer, and in most cases an orthokeratinized horny layer. The squamous metaplasias with severe atypia showed an increased secretory activity and a decreased number of desmosomes. Carcinomas in situ were characterized by numerous intracellular and extracellular microcysts filled with secretory material identified by histochemical techniques as containing neutral glycoproteins and sialomucins. Microinvasive carcinoma showed similar characteristics, with an increase in polylobulated nuclei and appearance of multilaminar ergastoplasm. An outstanding feature in all these lesions was the presence of dark fusiform epithelial cells. They were observed in the middle layers in the squamous metaplasias with severe atypia and appeared in all layers of the carcinomas in situ.

Quantitative assessment of generalized epithelial changes in tracheal mucosa following exposure to 7,12-dimethylbenz(a)anthracene.

Sequential morphological changes occurring after brief carcinogen exposures of heterotopic tracheal transplants in rats were semiquantitatively studied. Tracheas were exposed to 7,12-dimethylbenz(a)anthracene for 1, 2, or 4 weeks, during which time means of 138, 152, and 160 microgram 7,12-dimethylbenz(a)anthracene, respectively, were delivered. The first two types of exposures resulted only in generalized epithelial changes; these included hyperplasia and early metaplasia, both of which regressed rapidly, and persistent atrophic alterations. No focal epithelial lesions or tumors developed. The third type of exposure (160 microgram 7,12-dimethylbenz(a)anthracene delivered in 4 weeks) resulted in the appearance of generalized mucosal changes with long-lasting, severe inhibition of mucus production. In addition, focal metaplastic lesions reappeared at 4 to 8 months after exposure, and invasive carcinomas developed after 1 year with an incidence of 9%. Overall carcinoma incidence, including carcinoma in situ, was 15%. The studies emphasize the importance of the duration of carcinogen exposure, and they demonstrate the emergence of focal lesions when effective carcinogenic exposures are being used. The possible significance of epithelial atrophy in the pathogenesis of cancer in this experimental model is discussed.

Pathologic changes induced in respiratory tract mucosa by polycyclic hydrocarbons of differing carcinogenic activity.

Seven aromatic polycyclic hydrocarbons (PCHs) were investigated for their toxic effects on respiratory mucosa: benzo(e)pyrene (BeP), pyrene, anthracene, benz(a)anthracene(BaA), dibenz(a,c)anthracene(DBacA), benzo (a)pyrene (BaP), and dimethylbenz(a)anthracene (DMBA). The compounds were chosen because they comprise a spectrum of PCHs ranging from noncarcinogens, to initiators, to weak and strong carcinogens. All of them except DMBA are environmentally relevant chemicals. The chemicals were tested over an 8-week period. Heterotopic tracheal transplants were continously exposed and the histopathologic effects induced by the various PCHs were periodically assessed semiquantitatively. All PCHs exhibited varying degrees of toxicity for respiratory epithelium and submucosa. BeP clearly showed the least toxicity followed by pyrene and anthracene. BaA and DBacA caused marked epithelial and submucosal changes. In addition to epithelial hyperplasia, undifferentiated epithelium and squamous metaplasia developed. Marked mononuclear infiltration occurred in the subepithelial connective tissue. With BaP the epithelial and submucosal changes were similar but were much stronger. DMBA was the most toxic substance, causing epithelial necrosis followed by generalized keratinizing squamous metaplasia; the subepithelial changes consisted of an early acellular exudate and, later (at 8 weeks), marked condensation and hyalinization of the lamina propria. The toxic response pattern of the tracheal mucosa to carcinogenic agents was characterized by the chronicity of epithelial and connective tissue damage, as opposed to the short-lived hyperplastic and inflammatory response elicited by the noncarcinogens and weak initiators.

Synthesis of macromolecules by intestinal cells incubated with ammonia.

Mucosal cells isolated from the small intestine of chicks and rats were incubated with concentrations of ammonia normally found in the intestinal tract of mammals and birds. NH4Cl added to the incubation medium increased glucose metabolism in cells from both species. Ammonia stimulated incorporation of precursors into RNA and decarboxylation of orotic acid by cells isolated from chickens, but an increase in incorporation of precursors into DNA was not observed in cells from either species. Cultured embryonic chicken duodena showed increased incorporation of orotate into RNA with NH4Cl added to the medium. Rats immunized against jack bean urease showed lower urease activity per gram of dry intestinal content, lower intestinal weight, lower mucosal cell, and total gut protein and less protein per unit weight of DNA in the mucosal cell fraction. The results are compatible with the conclusion that ammonia PRODUCED IN THE INTESTINE BY BACTERIAL UREASES CAUSES SIGNIFICANT CHANGES IN THE CONTENT OF RNA and protein in intestine cells.