Experimental cryptorchidism enhances testicular susceptibility to dibutyl phthalate or acrylamide in Sprague-Dawley rats.

Cryptorchidism (CPT), the most common male congenital abnormality, is variably associated with other male reproductive tract problems. We evaluated if cryptorchid rats develop enhanced testicular susceptibility to dibutyl phthalate (DBP) or acrylamide (AA) after extended exposure. Three studies with rats were performed: (1) in utero and postnatal exposure to DBP or AA; (2) establishment of CPT and orchiopexy; and (3) in utero and postnatal exposures to DBP or AA associated with CPT/orchiopexy. Seminiferous tubules were histologically scored according to the severity of lesions: (1) Rats exposed to DBP (score 1.5) or AA (score 1.1) presented mostly preserved spermatogenesis. Some seminiferous tubules showed vacuolated germinative epithelium, germ cell apoptosis, and a Sertoli cell-only (SCO) pattern. (2) CPT (score 3.3) resulted in decreased absolute testes weights, degenerated and SCO tubules, and spermatogenesis arrest that were reversed by orchiopexy (score 1.1). (3) Exposure to DBP or AA with CPT/orchiopexy led to atrophic testes, spermatogenesis arrest, germ cell exfoliation/multinucleation, and SCO tubules (both chemicals score 2.5). Exposure to chemicals such as DBP or AA prevented the recovery of cryptorchid testes by orchiopexy. The possible role of environmental contaminants should be considered when looking for factors that modulate human testicular disorders associated with CPT.

Local adaptation for body color in Drosophila americana.

Pigmentation is one of the most variable traits within and between Drosophila species. Much of this diversity appears to be adaptive, with environmental factors often invoked as selective forces. Here, we describe the geographic structure of pigmentation in Drosophila americana and evaluate the hypothesis that it is a locally adapted trait. Body pigmentation was quantified using digital images and spectrometry in up to 10 flies from each of 93 isofemale lines collected from 17 locations across the United States and found to correlate most strongly with longitude. Sequence variation at putatively neutral loci showed no evidence of population structure and was inconsistent with an isolation-by-distance model, suggesting that the pigmentation cline exists despite extensive gene flow throughout the species range, and is most likely the product of natural selection. In all other Drosophila species examined to date, dark pigmentation is associated with arid habitats; however, in D. americana, the darkest flies were collected from the most humid regions. To investigate this relationship further, we examined desiccation resistance attributable to an allele that darkens pigmentation in D. americana. We found no significant effect of pigmentation on desiccation resistance in this experiment, suggesting that pigmentation and desiccation resistance are not unequivocally linked in all Drosophila species.

The effects of phenytoin on the performance of rats in a delayed match-to-place task.

We have shown previously that phenytoin impairs learning in rats in several different behavioral paradigms (Churchill et al., 1998, 2003; Banks et al., 1999). The present study has examined this drug's effects on performance in a delayed match-to-place water maze paradigm developed by Steele and Morris (1999). We find that phenytoin retards performance, but only when the inter-trial interval (ITI) is short (i.e., 15-sec). With longer ITIs (i.e., 20-min, 2-hr), the performance of the phenytoin-treated rats was quite comparable to the controls. We suggest that this pattern of results stems from a disruption of spatial working memory, perhaps due to the effects of the drug on hippocampal function (cf., Churchill et al., 1998, 2003). This disruption is, however, not so profound that consolidation is prevented.

The effects of estradiol on avoidance learning in ovariectomized adult rats.

The present study examined the effects of ovariectomy and subsequent estradiol replacement on learning in young adult rats using a set of instrumental avoidance paradigms differing in the nature and extent of prior experience in the learning context. Thus, one group of animals was placed directly into avoidance learning (AV). A second group was trained on an appetitive task first, and then transferred into the aversive context (AP-AV). The third group was exposed to the training context without any specific appetitive response requirement, and then required to learn an active avoidance response (Context-AV). We found that estradiol (OVX+E) impaired avoidance acquisition in all cases relative ovariectomized controls (OVX). In contrast, while avoidance learning is improved following appetitive training or context exposure in both OVX+E and OVX animals, the OVX+E animals profit to a greater extent from the appetitive or context experience than do the OVX controls. We suggest that this difference may be due to enhanced attentional processes or improved hippocampal processing of contextual factors. Thus, estradiol negatively influences simple associative avoidance learning in ovariectomized rats, but appears to promote positive transfer.

Lesions of the rat nucleus basalis magnocellularis disrupt appetitive-to-aversive transfer learning.

Rats with selective lesions of the nucleus basalis magnocellularis (NBM) and sham-lesion control animals were tested in an operant appetitive-to-aversive transfer task. We hypothesized that NBM lesions would not affect performance in the appetitive phase, but that performance would be impaired during subsequent transfer to the aversive phase of the task. Additional groups of NBM lesion and control rats were tested in the avoidance condition only, where we hypothesized that NBM lesions would not disrupt performance. These hypotheses were based on the argument that the NBM is not necessary for simple association learning that does not tax attention. Both the appetitive phase of the transfer task and the avoidance only task depend only on simple associative learning and are argued not to tax attention. Consequently, performance in these tasks was predicted to be spared following NBM lesions. Complex, attention-demanding associative learning, however, is argued to depend on the NBM. Performance in the aversive phase of the transfer task is both attentionally demanding and associatively more complex than in either the appetitive or aversive tasks alone; thus, avoidance performance in the NBM lesion group was predicted to be impaired following transfer from prior appetitive conditioning. Results supported our hypotheses, with the NBM lesion group acquiring the appetitive response normally, but showing impaired performance following transfer to the aversive conditioning phase of the transfer task. Impairments were not attributable to disrupted avoidance learning per se, as avoidance behavior was normal in the NBM lesion group tested in the avoidance condition only.

Acute reductions in GABAA receptor binding in layer IV of adult primate somatosensory cortex after peripheral nerve injury.

Following peripheral nerve transection, reorganizational plasticity has been reported to occur in two phases, one immediate and one more protracted. GABA (gamma-aminobutyric acid) has been implicated in the immediate "unmasking" phase of reorganization. We have used quantitative autoradiography to assess potential changes in GABA(A) and GABA(B) receptor binding in primate somatosensory cortex following peripheral nerve injury. Here we report reductions in GABA(A) receptor binding in layer IV of primate somatosensory cortex deprived of its normal activating inputs for 2-5 h by peripheral nerve transection.

Observed classroom behavior of children with ADHD: relationship to gender and comorbidity.

Examined hypothesized gender and comorbidity differences in the observed classroom behavior of children with attention deficit hyperactivity disorder (ADHD). The behavior of 403 boys and 99 girls with ADHD, ages 7-10, was compared (a) to observed, sex-specific classroom behavior norms, (b) by sex, and (c) by comorbid subgroups. Boys and girls with ADHD deviated significantly from classroom norms on 15/16 and 13/16 categories, respectively. Compared to comparison girls, girls with ADHD had relatively high rates of verbal aggression to children. Boys with ADHD engaged in more rule-breaking and externalizing behaviors than did girls with ADHD, but the sexes did not differ on more "neutral," unobtrusive behaviors. The sex differences are consistent with notions of why girls with ADHD are identified and referred later than boys. Contrary to hypothesis, the presence of comorbid anxiety disorder (ANX) was not associated with behavioral suppression; yet, as hypothesized, children with a comorbid disruptive behavior disorder (DBD) had higher rates of rule-breaking, and impulsive and aggressive behavior, than did children with ADHD alone and those with ADHD+ANX. Elevated rates of ADHD behaviors were also observed in children with comorbid DBD, indicating that these behaviors are truly present and suggesting that reports of higher ADHD ratings in this subgroup are not simply a consequence of negative halo effects and rater biases.

Urinary tract calculi and thresholds in carcinogenesis.

Numerous chemicals administered to rodents at relatively high doses produce urinary tract calculi, resulting in erosions or ulcerations of the urothelium, consequent regenerative hyperplasia, and ultimately tumors. This is a high-dose (threshold) phenomenon, which appears to occur more readily in rodents than in primates, including humans. Several anatomic and urinary physiologic differences between rodents and humans affect the quantitative extrapolation from results in rodent bioassays to human risk assessment. For most chemicals producing tumors by this mode of action, human exposures are significantly lower than would be expected to be required for production of calculi, and therefore pose no carcinogenic hazard to humans.

Somatotopic reorganization in the brainstem and thalamus following peripheral nerve injury in adult primates.

Injury-induced reorganization of central somatotopic maps is a phenomenon that has proven to be useful for elucidating the mechanisms and time course of neural plasticity. To date, the overwhelming majority of this line of research has focused on such plastic events in cortical areas, at the expense of subcortical structures. In this study, we used multi-unit electrophysiological recording techniques to assess the somatotopic organization of brainstem and thalamic areas following chronic survival from paired median and ulnar nerve section in adult squirrel monkeys. We report that the extent of cutaneously-driven reorganization in both the cuneate nucleus of the brainstem and the ventroposterior lateral nucleus of the thalamus is comparable to that previously documented for area 3b of cortex. These observations are consistent with those previously reported in thalamus, and are unique for brainstem.

Effect of sulfosulfuron on the urine and urothelium of male rats.

Sulfosulfuron, developed as a herbicide, caused increased microcrystalluria and the formation of urinary tract calculi when fed to male and female rats in a chronic 2-year study at doses of 5,000 ppm and 20,000 ppm. Hyperplasia was also seen in urinary bladders at 5,000 ppm and 20,000 ppm, almost exclusively in the presence of observable calculi/microcalculi. Urinary bladder tumors were found in 2 females in the 5000 ppm group, both in the presence of calculi. No increased microcrystalluria, calculi, or tumors were found at doses of 500 ppm and lower. In the current study, 5 groups of male Sprague-Dawley rats were fed sulfosulfuron at doses of 50, 500, 5,000, and 20,000 ppm for 10 weeks. Ten animals were co-administered 5,000 ppm sulfosulfuron with 12,300 ppm NH4Cl to determine if inhibition of the formation of calculi would prevent any urothelial effects of treatment with sulfosulfuron. Ten animals in the control group and in the high-dose sulfosulfuron group were fed only basal diet for an additional 10 weeks to determine if the effects of sulfosulfuron on the bladder epithelium were reversible. There was an increased incidence of microcrystalluria observed at 5,000 and 20,000 ppm. There was no increase in microcrystalluria observed in the urine of rats co-administered sulfosulfuron and NH4Cl. Urinary bladder calculi were found in the bladder of 1 animal fed 20,000 ppm. Examination by light microscopy showed diffuse papillary/nodular hyperplasia of the bladder epithelium in this animal. No increased microcrystalluria was observed after withdrawal of the chemical from the diet and the bladder epithelium was normal by light microscopy. The hyperplastic effects associated with the feeding of high doses of sulfosulfuron occur only with the appearance of urinary tract calculi. Based on these results and anatomical differences between rats and humans, it may be concluded that the hyperplastic and carcinogenic effects of sulfosulfuron in rats are high-dose, threshold phenomena that are not likely to occur in humans under environmentally relevant exposures.

Dietary effects of ortho-phenylphenol and sodium ortho-phenylphenate on rat urothelium.

Ortho-phenylphenol (OPP) and sodium ortho-phenylphenate (NaOPP) are pesticides used commercially in the food industry that have been shown to be carcinogenic to rat urothelium. Dietary administration of 1.25% OPP or 2.0% NaOPP caused increased incidences of urothelial hyperplasia and eventually caused tumors in male F344 rats, with NaOPP apparently having a more potent effect. In other studies, various sodium salts such as saccharin and ascorbate enhanced bladder carcinogenesis, although the acid forms of these salts did not. In studies with high dietary doses of these sodium salts, an amorphous precipitate was produced in the urine; precipitate formation was pH dependent. In previous experiments in which high doses of OPP were fed for up to 17 weeks, severe hyperplasia of the urothelium was produced, but without the formation of an urinary amorphous precipitate, calculi, or abnormal microcrystalluria. In addition, we found no evidence of OPP-DNA adduct formation in the urothelium. The present study was conducted to determine if feeding NaOPP * 4 H(2)0 to male F344 rats as 2.0% of the diet resulted in the formation of an amorphous precipitate in the urine, and if NaOPP caused an increased mineral concentration in the urine and/or kidneys. NaOPP administration produced a higher urinary pH than did OPP fed as 1.25% of the diet. Neither amorphous precipitate nor other solids were observed in the urine of the OPP or NaOPP-treated rats, and urinary calcium concentrations in the treated groups were similar to control. OPP and NaOPP had similar proliferative effects on rat urothelium after 10 weeks of treatment by light microscopy, scanning electron microscopy (SEM), and bromodeoxyuridine (BrdU) labeling indices. The results of this study indicate that formation of abnormal urinary solids is not part of the mechanism by which OPP or NaOPP exert their effects on the rat bladder epithelium.

Urothelial cytotoxicity and regeneration induced by dimethylarsinic acid in rats.

Inorganic arsenic is a known human carcinogen of the skin and respiratory tract. Epidemiologic evidence indicates that it is also carcinogenic to the urinary bladder and other internal organs. Lack of an animal model has limited progress on understanding the mechanism of arsenic carcinogenesis. It was recently reported that high doses of an organic arsenical, dimethylarsinic acid (DMA), increased urinary bladder tumors in rats when administered in the diet or in the drinking water for 2 years, with the female being more sensitive than the male. We previously showed that high doses of DMA (40 or 100 ppm of the diet) fed for 10 weeks increased urothelial cell proliferation in the rat. Treatment with DMA also increased renal calcification and increased urinary calcium concentration. In 2 experiments, we examined the urothelial proliferative effects of treatment with 100 ppm DMA in the diet in female F344 rats for 2 and 10 weeks and for 6 and 24 h, and 3, 7, and 14 days. Cytotoxic changes in the urothelium were evident by SEM as early as 6 h after treatment was begun. Foci of cellular necrosis were detected after 3 days of treatment, followed by widespread necrosis of the urothelium after 7 days of treatment. The bromodeoxyuridine (BrdU) labeling index was not increased until after 7 days of treatment, suggesting that administration of DMA results in cytotoxicity with necrosis, followed by regenerative hyperplasia of the bladder epithelium. Although the rat provides an animal model to study the urothelial effects of DMA, the relevance of this finding to inorganic arsenic carcinogenesis in humans must be extrapolated cautiously, due to the high doses of DMA necessary to produce these changes in the rat and the differences in metabolism of arsenicals in rodents, especially rats, compared to humans.

Evaluation of diet and dimethylarsinic acid on the urothelium of Syrian golden hamsters.

Few studies have examined the carcinogenicity of chemicals toward the urinary bladder in hamsters, and the effect of diet on hamster urine and urothelium has not been reported. Our laboratory recently began investigating the effects of dimethylarsinic acid (DMA) on the hamster bladder, and we noticed subtle urothelial changes even in controls. The possible effect of various diets on hamster urothelium was evaluated by feeding different diets to 4-week-old Syrian Golden hamsters for 5 weeks. The diets examined were Tekland 8656, Purina 5002, Purina 5L79, and NIH-07. Light microscopic examination showed a slight increase in urothelial hyperplasia in hamsters fed Purina 5L79. An increase in the incidence of urinary bladder necrosis, exfoliation, and mild hyperplasia were noted by scanning electron microscopy (SEM) with all dietary preparations except NIH-07. The constituents in the diets producing the urothelial alterations are not known at present, but NIH-07 diet was chosen for experiments to investigate the effects of DMA on the hamster bladder epithelium. Male and female 5-week-old Syrian Golden hamsters were fed 100 ppm DMA for 10 weeks. Examination of urinary parameters showed no treatment-related changes. Light microscopic examination and SEM revealed no changes of the urothelium of DMA-treated male or female hamsters.

Calcium phosphate-containing precipitate and the carcinogenicity of sodium salts in rats.

Sodium saccharin, ascorbate and other sodium salts fed at high doses to rats produce urinary bladder urothelial cytotoxicity with consequent regenerative hyperplasia. For sodium salts that have been tested, tumor activity is enhanced when administered either alone or after a brief exposure to a known genotoxic bladder carcinogen. These sodium salts alter urinary composition of rats resulting in formation of an amorphous precipitate. We examined the precipitate to ascertain its composition and further delineate the basis for its formation in rat urine. Using scanning electron microscopy with attached X-ray energy dispersive spectroscopy, the principal elements present were calcium, phosphorus, minor amounts of silicon and sulfur. Smaller elements are not detectable by this method. Infrared analyses demonstrated that calcium phosphate was in the tribasic form and silicon was most likely in the form of silica. Small amounts of saccharin were present in the precipitate from rats fed sodium saccharin (<5%), but ascorbate was not detectable in the precipitate from rats fed similar doses of sodium ascorbate. Large amounts of urea and mucopolysaccharide, apparently chondroitin sulfate, were detected in the precipitate by infrared analysis. Chemical analyses confirmed the presence of large amounts of calcium phosphate with variably small amounts of magnesium, possibly present as magnesium ammonium phosphate crystals, present in urine even in controls. Small amounts of protein, including albumin and alpha(2u)-globulin, were also detected (<5% of the precipitate). Calcium phosphate is an essential ingredient of the medium for tissue culture of epithelial cells, but when present at high concentrations (>5 mM) it precipitates and becomes cytotoxic. The nature of the precipitate reflects the unique composition of rat urine and helps to explain the basis for the species specificity of the cytotoxic and proliferative effects of high doses of these sodium salts.

Effects of dietary dimethylarsinic acid on the urine and urothelium of rats.

Dimethylarsinic acid (DMA), fed to rats for 2 years, produced bladder hyperplasia and tumors at doses of 40 and 100 p.p.m., more in females than males. No urothelial proliferation was seen in mice. Our objectives were to investigate the mode of action of bladder tumor formation, evaluate the dose-response and the role of diet and to determine if the urothelial effects were reversible. The study included groups of female F344 rats fed DMA in Purina 5002 diet at doses of 0, 2, 10, 40 or 100 p.p.m. for 10 weeks; two groups of females fed DMA (0 and 100 p.p.m.) in Altromin 1321 for 10 weeks; two groups of males fed DMA (0 and 100 p.p.m.) in Purina 5002 for 10 weeks; a female high-dose recovery group (100 p.p.m. in Purina 5002 diet for 10 weeks followed by control diet for 10 weeks); and two female groups (0 and 100 p.p.m.) in Purina diet for 20 weeks. Urothelial toxicity and hyperplasia were detected by light and scanning electron microscopy (SEM), and the bromodeoxyuridine labeling index was increased in the female 40 and 100 p.p.m. groups. The effects were less in males, but were similar in females fed DMA in Altromin 1321. SEM detected no abnormal urinary solids related to treatment in any group. Urinary calcium was increased in the females fed 40 and 100 p.p.m. in Purina diet, despite overall urinary dilution. Calcification was increased in kidneys of female rats fed Purina diet. The urothelial effects of DMA were reversible. The findings support a non-DNA reactive mechanism for DMA rat bladder carcinogenicity related to urothelial toxicity and regeneration. The toxicity is probably not due to urinary solids. The toxicity and regeneration are produced in a dose-responsive manner in female rats, are greater in female than in male rats, and are reversible.

Pregnancy-specific glycoprotein gene expression in recurrent aborters: a potential correlation to interleukin-10 expression.

PROBLEM: The expression of the pregnancy-specific glycoprotein (PSG) genes in the uterine endometrium of women experiencing recurrent first-trimester abortions, and potential correlations to cytokine expression were examined. METHOD OF STUDY: Endometrial RNA, isolated from women with a history of either repetitive first-trimester pregnancy losses or uncomplicated pregnancies, was isolated and analyzed for PSG transcripts by the reverse transcriptase-polymerase chain reaction method. PSG genes showing different patterns of expression were expressed in baculovirus, and the purified proteins examined for their effects on cytokine expression. RESULTS: The expression of PSG11 in the endometria of recurrent aborters was significantly lower than in that of controls (P < 0.01). When tested on monocytes, PSG11 stimulated secretion of interleukin (IL)-10. CONCLUSIONS: The level of expression of the PSG11 gene in the uterine endometrium, during the peri-implantation period, correlates with the risk of pregnancy loss in some women experiencing recurrent spontaneous abortions. The ability of PSG11 to influence the secretion of IL-10 suggests that PSG11 may contribute to the local modulation of the inflammatory T helper-1 response seen in the endometrium of these women.

Effect of pregnancy-specific beta 1-glycoprotein on the development of preimplantation embryo.

The objective of this study is to test the hypothesis that members of the pregnancy-specific beta 1-glycoprotein (PSG) family enhance the growth and maturation of embryos. cDNA encoding two members of the PSG family, namely PSG1 and PSG3, were expressed in Chinese Hamster Ovary (CHO) cells with the expression vector pH beta APr-1-neo. Two-cell stage mouse embryos were co-cultured in a two-chamber system with CHO cells expressing either recombinant PSG1 (rPSG1) or PSG3 (rPSG3) in the presence and absence of neutralizing PSG antibodies. The cleavage and maturation stage of the embryos was assessed at 12-hr intervals. Mouse embryos co-cultured with transfectants expressing rPSG1 showed a significant enhancement of cleavage and maturation rate compared to controls with P < 0.005-0.004. In co-cultures with CHO cells expressing rPSG3, no significant difference from the controls was observed in the early stage of development until late blastocyst formation. At that stage, there was a statistically significant enhancement of development by rPSG3 when compared to controls with P < 0.001. These results suggest that PSG1 and PSG3 exhibit embryotropic activity at different stages of development in the mouse model.

Urinary physiologic and chemical metabolic effects on the urothelial cytotoxicity and potential DNA adducts of o-phenylphenol in male rats.

ortho-Phenylphenol (OPP), a fungicide and antibacterial agent with food residues, is carcinogenic to rat bladder. The present studies provide information on changes in urinary composition and urinary metabolites, urothelial cytotoxicity and regenerative hyperplasia, and DNA adducts in male F344 rats fed OPP. An initial experiment evaluated dietary doses of 0, 1,000, 4,000, and 12,500 ppm OPP fed for 13 weeks. There was no evidence of urinary calculi, microcrystalluria, or calcium phosphate-containing precipitate, but urothelial cytotoxicity and hyperplasia occurred at the highest dose only. In a second experiment, rats were fed dietary OPP levels of 0, 800, 4,000, 8,000, and 12,500 ppm. Urinary pH was > 7 in all groups. Urinary volume was increased at the 2 highest doses with consequent decreases in osmolality, creatinine, and other solutes. Total urinary OPP metabolite excretions were increased, mostly excreted as conjugates of OPP and of phenylhydroquinone. Free OPP or free metabolites accounted for less than 2% excreted in the urine without a dose response. Urothelial toxicity and hyperplasia occurred only at doses of 8,000 and 12,500 ppm. OPP-DNA adducts were not detected in the urothelium at any dose. In summary, OPP produces cytotoxicity and proliferation of the urothelium at dietary doses > or = 8,000 ppm without formation of urinary solids. The paucity of unconjugated metabolites and the lack of OPP-DNA adducts suggests that OPP is acting as a bladder carcinogen in male rats by inducing cytotoxicity and hyperplasia without it or its metabolites directly binding to DNA.

Tumorigenicity of sodium ascorbate in male rats.

Sodium ascorbate, like other sodium salts such as saccharin, glutamate, and bicarbonate, produces urinary alterations when fed at high doses to rats, which results in mild superficial urothelial cytotoxicity and regeneration but not tumors in a standard 2-year bioassay. Sodium saccharin was shown to produce a low incidence of bladder tumors in rats if administered in a two-generation bioassay. In the present study, we evaluated sodium ascorbate in a two-generation bioassay that involved feeding to the male and female parental F344 rats for 4 weeks before mating, feeding the dams during gestation and lactation, and then feeding the weaned (at 28 days of age) male F1 generation rats for the remainder of their lifetime (up to 128 weeks of the experiment). Dietary levels of 1.0, 5.0, and 7.0% sodium ascorbate were tested. At 5.0 and 7.0% sodium ascorbate, there was an increase in urinary bladder urothelial papillary and nodular hyperplasia and the induction of a few papillomas and carcinomas. There was a dose-responsive increase in renal pelvic calcification and hyperplasia and inhibition of the aging nephropathy of rats even at the level of 1% sodium ascorbate. Because the short-term urothelial effects of sodium ascorbate in rats are inhibited by treatments producing urinary acidification to pH < 6.0, we coadministered high doses of long-term NH4Cl to groups of rats with 5.0 or 7.0% sodium ascorbate to evaluate the long-term effects. The combination of 7.0% sodium ascorbate plus 2.78% NH4Cl in the diet was toxic, and the group was terminated early during the course of the experiment. The group fed 5.0% sodium ascorbate plus 2.04% NH4Cl showed complete inhibition of the urothelial effects of sodium ascorbate and significant inhibition of its renal effects. We also demonstrated the presence of a calcium phosphate-containing urinary precipitate in rats fed sodium ascorbate at all doses, in a dose-responsive manner. The formation of the precipitate was inhibited by coadministration with NH4Cl. The proliferative effects of sodium ascorbate on the male rat urinary tract in this study are similar to those seen with sodium saccharin when administered in a two-generation bioassay. Mechanistic information suggests that this is a high-dose, rat-specific phenomenon.