Hyaline Droplets Research Articles

Lysosomal Changes in Renal Proximal Tubular Epithelial Cells of Male Sprague Dawley Rats Following Decalin Exposure

A histochemical stain for acid phosphatase served as a marker for lysosomal alterations in renal tubular cells associated with male rat hyaline droplet nephropathy. Morphometric analysis and quantitative histochemistry were used to compare the size and acid phosphatase stain reaction of lysosomes in tubular epithelial cells of treated and control animals. Decalin exposure increased the size and significantly (p less than 0.01) reduced the acid phosphatase stain intensity of individual lysosomes. However, there was no significant different (p greater than 0.05) between the acid phosphatase stain intensity of treated and control animals when analyzed on a whole cell basis. The increase in size of the lysosomes without a proportional increase in whole cell acid phosphatase stain intensity indicates a dilution or a failure to accommodate in the acid phosphatase concentration (stain intensity/microns 2) per lysosome. All acid phosphatase stain reaction product was contained within intact lysosomes, mitigating against the hypothesis of lysosomal enzyme leakage as the cause of cell death in decalin-induced alpha 2U globulin nephropathy.

Lysosomal degradation of α2u-globulin and α2u-globulin-xenobiotic conjugates

A diverse group of chemicals cause a male rat-specific nephrotoxicity in which α2u-globulin accumulates in renal lysosomes. It has been suggested that these chemicals bind to the protein and decrease its degradation by lysosomal proteinases. To test this hypothesis, the lysosomal degradation of native α2u-globulin and that to which d-limonene, d-limonene-1,2-oxide, isophorone, 1,4-dichlorobenzene, and 2,5-dichlorophenol were bound was studied. α2u-Globulin was purified from male rat urine, and male rat renal cortical lysosomes, isolated by differential centrifugation, served as the proteolytic enzyme source. Pepstatin, an inhibitor of aspartic acid proteinases, and leupeptin, an inhibitor of cysteine proteinases, reduced α2u-globulin degradation to 28 ± 8 and 17 ± 5% of control, respectively, whereas addition of both inhibitors decreased α2u-globulin degradation to 8 ± 1% of control values. These results indicate that both classes of endopeptidases are important in the degradation of α2u-globulin. Under the incubation conditions used, 30% of native α2u-globulin was degraded in a 4-hr period. Conjugates of the protein were made for in vitro binding experiments. Binding of d-limonene and 1,4-dichlorobenzene to α2u-globulin did not alter the degradation of the protein, whereas binding of d-limonene-1,2-oxide, 2,5-dichlorophenol, and isophorone decreased α2u-globulin degradation by 33%. These results indicate that not all chemicals which have been shown to bind in vivo to α2u-globulin alter the in vitro lysosomal degradation of the protein. However, in all cases, one metabolite of each hyaline droplet inducer did alter degradation of α2u-globulin, suggesting that a decrease in lysosomal degradation is involved in the accumulation of this protein in male rat kidney lysosomes.

A comparison of male rat and human urinary proteins: Implications for human resistance to hyaline droplet nephropathy

α 2u-Globulin (αG), the major urinary protein of sexually mature male rats, is a key determinant of susceptibility to hyaline droplet nephropathy (HDN) induced by a variety of hydrocarbons in male rats. Arguments against extrapolating renal toxicity and carcinogenicity data for HDN-inducing toxicants from male rats to risk assessment for humans rely on the observation that humans do not express αG. Yet, human serum and urine are known to contain proteins coded for by the same gene family that also controls αG synthesis in the rat. Therefore, to understand some of the quantitative and qualitative differences between proteins of human and male rat urine which confer apparent resistance to HDN in humans, urinary proteins of male F344 rats (ca. 3 months old) and normal human males were compared by cation exchange, gel filtration, SDS-PAGE, and partially identified by Western blotting. We observed that (1) the protein content of human urine is only 1% that of male rat urine; (2) human urinary proteins, recovered by (NH 4) 2SO 4 precipitation followed by dialysis, are primarily of high (≥75 kDa) molecular weight (MW) with minor components of 12–66 kDa; (3) male rat urine has little high-MW protein, but is rich in αG (18.5 kDa); (4) at pH 5, the most cationic fraction of human urinary protein constituted only about 4% of the total while the analogous fraction of rat urine, containing αG, contained 26% of total urinary protein; and (5) cationic (at pH 5.0) human urinary proteins included small amounts of proteins, e.g., α 1-acid glycoprotein, and α 1-microglobulin, which are products of the gene family coding for αG in rat. Thus, although humans excrete trace amounts of proteins similar to αG, the very low protein content of human urine, the relatively small proportion of cationic to total proteins, and the high MW of the most abundant human urinary proteins form a biological basis for suggesting that humans are not at risk for the type of fuel and solvent hydrocarbon-induced nephropathy, and the sequelae of such nephropathy, observed in male rats.

Assessment of the subchronic oral toxicity of d-limonene in dogs

Several hydrocarbons, including d-limonene, have been shown to produce a male-rat-specific nephrotoxicity that is manifested acutely as exacerbation of hyaline droplet formation. In a study to assess the presence or absence of this response in a non-rodent species, the dog was selected as a relevant model because of an earlier report suggesting that d-limonene may be nephrotoxic in this species. Five male and five female adult beagle dogs per treatment group were gavaged twice daily over a 6-month period with tap-water (control) or d-limonene at 0.12 or 1.2 ml/kg body weight/day (100 or 1000 mg/kg body weight/day). The highest daily dose was determined in a pilot study to be close to the maximum tolerated dose for emesis (ED 50 1.6 ml/kg body weight). The test compound was administered in divided doses to minimize the incidence of emesis. Feed consumption and body weight were unaffected by treatment. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining, that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation nor of any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene. Thus, dogs are refractory to the hyaline droplet nephropathy observed in male rats, thereby providing additional evidence that the male rat kidney is uniquely sensitive to hydrocarbons like d-limonene, and that this specific male rat nephropathic response may be inappropriate for interspecies extrapolation and human risk assessment.

Subchronic Inhalation Studies on Polychlorotrifluoroethylene (3.1 Oil)

Abstract3.1 Oil, an oligomer of chlorotrifluoroethylene (CTFE), is an inert, nonflammable, saturated and hydrogen-free chlorofluorocarbon oil of interest to the Department of Defense (DOD) as a potential hydraulic fluid. To determine the potential subchronic inhalation toxicity of this CTFE oligomer, male and female F-344 rats were exposed to air only or to 0.25, 0.50, or 1.00 mg/l of CTFE oligomer in 65 6-h inhalation exposures over a 90-day period. A dose-dependent depression in body weight gains was noted in male rats only Serum alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities examined at the conclusion of the study indicated a treatment-related effect in the male test rats, but not the female test rats. Notable concentration-related increases (p < .07) in relative kidney and liver weights occurred in both sexes of rats at all test concentrations. The male rats showed slight to minimal hyaline droplet formation in the kidney proximal tubule epithelium. Pronounce...

Increased Hyaline Droplet Formation in Male Rats Exposed to Decalin Is Dependent on the Presence of α2u-Globulin

Increased Hyaline Droplet Formation in Male Rats Exposed to Decalin Is Dependent on the Presence of α_2u-Globulin

The effects of lindane and linuron on calcium metabolism, bone morphometry and the kidney in rats

Experiments were performed to investigate the effects of lindane and linuron on calcium metabolism, femur morphometry and nephrotoxicity. Long-Evans hooded rats were dosed daily for 10 weeks with 0, 10 or 20 mg lindane or 10, 20 or 40 mg linuron/kg body weight beginning at weaning. Lindane significantly decreased urinary calcium concentration, serum alkaline phosphatase concentration and the cross-sectional medullary area of the bone. Lindane was nephrotoxic at both dose levels as demonstrated by elevated kidney weights, kidney-to-body-weight ratios, urinary LDH, tubule regeneration and hyaline droplet degeneration. Linuron significantly reduced medullary cross-sectional area at the 2 higher dose levels and decreased the total femur cross-sectional area at the highest dose level in the absence of effects on calcium excretion. Femur density and strength were also significantly reduced at the highest dose level of linuron. Neither compound affected the serum concentrations of parathyroid hormone or 1,25-dihydroxy Vitamin D-3. Both linuron and lindane exposure significantly increased serum cholesterol concentrations and reduced serum triglyceride concentrations. Both compounds affected calcium metabolism and/or bone morphometry but possibly by different mechanisms since the effects were not the same.

Acute and subchronic nephrotoxicity of d-limonene in fischer 344 rats

In the studies described here, we have examined the sex-specific sensitivity of rat kidney to d-limonene. At 24 hr after an acute dose of 200 mg d-limonene/kg body weight administered to adult male and female Fischer 344 rats by oral gavage, an increase in the incidence and severity of hyaline droplets was observed in the kidneys of males only. This histological change was accompanied by a treatment-related increase in alpha 2u-globulin in males only and a greater accumulation of radioactivity in renal cortex of the male rat compared with that in the females dosed with [ 14 C]d- limonene . In a separate subchronic study, groups of 5-wk-old male rats were administered d-limonene in a corn oil vehicle at 0 (control), 2, 5, 10, 30 or 75 mg/kg body weight by single daily gavage (5 days/wk) for 13 wk. Rats from selected dose groups received interim necropsies from days 8–29, while all groups were necropsied at the end of the study. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg d-limonene/kg body weight. Histological examination of kidney tissue confirmed that d-limonene induced changes characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes collectively classified as chronic nephrosis. The no-observable-effect level for these effects was 5 mg d-limonene/kg body weight. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose. It is concluded that treatment with d-limonene caused an increase in the formation of hyaline droplets in male rats only, that this increase was associated with an accumulation of alpha 2u-globulin, that d-limonene (or its metabolite) accumulated significantly in male rat kidney compared with that in females and that subchronic dosing produced a triad of morphological changes in the male rat kidney. These observations suggest that d-limonene caused nephrotoxicity specific to the male rat and that this toxicity may not be predictive of a similar response in humans.

Phagolysosomal alterations induced by unleaded gasoline in epithelial cells of the proximal convoluted tubules of male rats: effect of dose and treatment duration.

Short-term oral administration of unleaded gasoline to male rats reproduces the accumulation of phagolysosomes (hyaline droplets) in epithelial cells of the renal proximal convoluted tubules (PCT) observed following long-term inhalation of wholly volatilized gasoline. Phagolysosomes are partially composed of alpha 2u-globulin, a low-molecular-weight protein, unique to male rats. In this study, dose-dependent and chronologic alterations of phagolysosomes caused by gasoline were observed by transmission electron microscopy. Exposure to commercially available unleaded gasoline (0.4-2.0 ml/kg, po, once daily, 9 d) increased the number and size of phagolysosomes in epithelial cells of the PCT in male rat kidney. However, administration of 0.04 ml gasoline/kg or less was ineffective in inducing phagolysosomal accumulation. Subcellular analysis revealed that many of the phagolysosomes observed in treated rats (doses greater than 0.4 ml/kg) were angular and had cross-sectional diameters varying from 0.5 to 9 microns; in controls the majority of phagolysosomes were round and their diameter varied from 0.5 to 2.5 microns. Treatment of male rats with gasoline (2.0 ml/kg body weight, po, 1-9 d) caused a progressive increase in the number and size of phagolysosomes in PCT epithelial cells dependent on treatment duration. Alterations in phagolysosomal morphology and quantity occurred within 20 h following a single dose of gasoline, emphasizing that the process of phagolysosome accumulation is a dynamic phenomenon. Many of the enlarged phagolysosomes contained a condensed, crystalline core of greater electron density than the surrounding matrix. Furthermore, the rapid increase in abnormal, condensed contents in the phagolysosomes may indicate that a derangement of renal protein catabolism is the primary mechanism by which fuel hydrocarbons cause hyaline droplet nephropathy in male rats.

The effect of the elastase on aminonucleoside nephrosis

In this paper, we studied the effect of elastase on aminonucleoside (AN) nephrosis which is considered a model of focal glomerular sclerosis (FGS). Elastase is an enzyme which disintegrates elastin, discovered by Balo, and used in the treatment of arteriosclerosis and hyperlipidemia. It has also been known to improve metabolism of acid mucopolysaccharides, so, this study focused on metabolic improvement. Three groups of male Sprague-Dawley rats were studied and observed at regular intervals; 30, 60, 90 days. The ANE group (AN + elastase) was administered one shot of AN (10 mg/100 g B.W.) during the test interval, while elastase (5 mg/kg B.W.) was injected 5 days/week for the entire test interval. The AN group was administered one shot of AN only. The third group was a control (C). The following results were observed: (1) Focal segmental hyalinosis and sclerosis (FSHS); ANE group was weaker than AN group. (2) Other significant qualifying glomerular changes (vacuolar change and hyaline droplets of the epithelial cells, adhesion, and foam cells); ANE group was weaker than AN group. (3) Anion loss in GBM was shown by a lack of colloidal iron staining under light microscopy, and by a lack of PEI particles under electron microscopy; there was significantly less anion loss with ANE group, than with AN group. The findings suggest that elastase has an affect on the metabolism of acid mucopolysaccharide and collagen in sclerotic lesion, and may restrain the progress of amino-nucleoside nephrosis.

Nutritional and pathological changes in male and female rats fed modifications of the AIN-76A diet

Male and female weanling Sprague-Dawley rats were fed either an AIN-76A diet or a modification of the AIN-76A diet containing no added dl-methionine but with higher levels of vitamins, fluoride and magnesium than in the AIN-76A diet. Both diets were fed, to groups of ten rats of each sex, at 18% protein or a reduced protein level of 13% for 12 wk. Within each sex, all diets produced comparable weight gains in rats at the end of 12 wk, except that the reduced-protein modified AIN-76A diet was associated with a reduction in weight gain in male rats. Both diet and protein level had statistically significant effects on the relative weights of some organs, particularly the kidney. The AIN-76A and the reduced-protein AIN-76A diets significantly increased the relative kidney weights (% body weights) of female rats, when compared with the effects of both modified AIN-76A diets (18 and 13% protein). Male rats fed both of the diets containing 18% protein had higher relative kidney weights than did those consuming both 13% protein diets. Females fed the modified diet containing 13% protein had significantly lower liver weights than the other groups. In both sexes, the two diets containing 18% protein caused significantly higher plasma urea nitrogen concentrations than did the lower protein diets. Kidney calcium concentrations varied with the diet, with dietary protein level, and with the sex of the animal. All diets caused small mineral (calcific) concretions of minimal to mild severity in the lumina of scattered renal tubules in the cortex and/or medulla of male rats. All female rats fed the AIN-76A and the reduced-protein AIN-76A diet had large, moderate or severe mineral concretions in the tubules at the corticomedullary junction and this was associated with increased renal calcium levels. The higher concentration of renal calcium at the lower dietary protein level (13%) was associated with severe corticomedullary junction mineralization. The higher protein diets were associated with an increased incidence of hyaline droplets in the cytoplasm of kidney cortical tubules in male rats.

Hydrocarbon-induced hyaline droplet nephropathy in male rats during senescence

Male rats administered unleaded gasoline rapidly develop nephropathy characterized by accumulation of hyaline droplets in cells of the proximal convoluted tubules (PCT). This acute response is implicated in development of renal carcinoma in male rats exposed chronically to wholly volatilized gasoline. A major constituent of hyaline droplets is α 2u-globulin, a protein of hepatic origin for which the rate of synthesis declines during aging. Little information, however, is presently available on possible age-dependent susceptibility of male rats to hydrocarbon-induced nephropathy. In kidneys of untreated male Fischer 344 rats the number of constitutive hyaline droplets declined progressively with increasing age. Electrophoresis of renal cortical homogenates revealed a protein with M r about 18 × 10 3, probably α 2u-globulin, in young (3.5 months old) male rats and total absence of this protein in aged (26 months old) males. RIA confirmed that constitutive levels of renal and hepatic α 2u-globulin in old rats were less than 1.5% of those in young adults. Unleaded gasoline (0.4 ml/kg/day, po, 5 days) caused accumulation of hyaline droplets in renal PCT of 3.5-month-old males accompanied by a marked increase (about twofold) in the renal content of α 2u-globulin, whereas the same treatment was without effect in 26-month-old rats. Finally, in the renal cortex of young rats the activities of the lysosomal proteases cathepsin B and D were increased following gasoline administration, presumably in response to protein accumulation. However, in 26-month-old rats cathepsin B activity was unaffected, while cathepsin D was increased by gasoline administration. Thus, we conclude that animal age is an important determinant in the development of hydrocarbon-induced nephropathy and only rats which produce large amounts of α 2u-globulin are susceptible to development of this pathology.

Induction and exacerbation of hyaline droplet formation in the proximal tubular cells of the kidneys from male rats receiving a variety of pharmacological agents

Kidneys from male and female Wistar rats dosed with 1 of 3 chemically unrelated pharmacological agents, a pyrazoline BW540C, a napthoquinone BW58C and the levoisomer of tetramisole, levamisole or the light hydrocarbon Decalin ®, were examined by light and electron microscopy. Paraffin histology showed that all 4 agents induced and exacerbated hyaline droplet accumulation in the renal proximal tubular cells of the male rats. Resin histology at both the light and electron microscope level, along with cytochemical procedures for acid phosphatase and the protein ‘alpha 2U globulin’, helped further in the characterisation of these cytoplasmic inclusions. These techniques confirmed that the accumulation of hyaline droplets seen by paraffin histology represented an increase in the size and number of secondary lysosomes which have been shown to be involved in protein uptake and metabolism. Time course studies showed that increased numbers of small dense lysosomes appear first, which then increase in size, presumably by fusion. Crystalloid bodies form in these large lysosomes eventually giving rise to rectilinear bodies. The accumulation of these protein laden secondary lysosomes took place primarily in the cells of the S 1 and S 2 segments of the proximal tubules. In extreme cases of lysosomal accumulation however, loading of the S 3 segments was noted. In tubules where cellular inclusion loading was heavy, there was evidence of increased cell turnover. The kidneys of female rats dosed with any one of the 4 agents appeared normal.

An immunohistochemical study of primary ovarian sarcoma. An evaluation of nine tumors.

Nine cases of primary ovarian sarcoma were studied with a panel of 10 antibodies by using the avidin-biotin-complex immunoperoxidase technique. Original diagnoses included five mixed müllerian tumors (MMT) homologous type, two MMT heterologous type, and two pure sarcomas (PS). Cytokeratin immunoreactivity was observed in epithelial components of all MMTs, but was not observed in PS. Two tumors originally classified as MMT also showed cytokeratin positivity in spindle-cell components. All the PSs and the spindle-cell components in six of seven MMTs showed positive staining for vimentin. Positive staining for desmin and myoglobin was found in one MMT in which cells with cytoplasmic cross-striations were identified on hematoxylin and eosin sections. However, less differentiated rhabdomyoblastic cells also stained for desmin and myoglobin, facilitating their recognition. Immunoreactivity for epithelial membrane antigen was noted in well-differentiated epithelial components of six MMT cases. One case stained with antibodies to carcinoembryonic antigen, four with S-100 protein, five with alpha-1-antitrypsin, and four with muramidase. No staining of stromal hyaline droplets was observed with any antibodies. Because of the immunohistochemical staining pattern, two of the original diagnoses were changed. Application of immunohistochemistry to spindle tumors of ovary is discussed.

Rapid postexposure decay of α2u‐globulin and hyaline droplets in the kidneys of gasoline‐treated male rats

Unleaded gasoline induces nephropathy, characterized by rapid accumulation of hyaline (protein resorption) droplets in epithelial cells of the renal proximal convoluted tubules, only in male rats. The hepatic synthesis of the male rat-specific protein alpha 2u-globulin, a constituent of renal hyaline droplets, is unaltered by gasoline treatment (Olson et al., 1987). Renal alpha 2u-globulin content increased to 210% of control within 18 h of a single oral dose of gasoline (2.0 ml/kg); maximal levels (320% of control) were attained following gasoline administration for 3 d. Increases in renal alpha 2u-globulin caused by gasoline were accompanied by concurrent proliferation of hyaline droplets. However, within 3 d of terminating gasoline administration renal alpha 2u-globulin content decreased to the same level as that in unexposed rats, although renal hyaline droplet number returned to pretreatment levels somewhat more slowly. The conjoint effect of postexposure recovery and estradiol (an inhibitor of hepatic alpha 2u-globulin synthesis) administration was also determined in male rats. On postexposure d 3, 6, and 9, estradiol treatment (1 mg/kg, sc, 4 d, starting on d 9 of gasoline treatment) decreased renal alpha 2u-globulin content to 75%, 59%, and 48%, respectively, of that in rats allowed to recover from gasoline with no hormone treatment. Hepatic alpha 2u-globulin content in estradiol-treated rats was decreased by 74%, 97%, and 96% at the same intervals. Estradiol treatment during recovery from gasoline also appeared to increase the removal of accumulated hyaline droplets from the renal cortex. Thus, accumulation of alpha 2u-globulin-containing hyaline droplets after subacute exposure of male rats to gasoline is rapidly reversible, dependent on continuous exposure to gasoline and maintenance of the normal rate of hepatic alpha 2u-globulin synthesis. These results emphasize the dynamic state of renal cortical hyaline droplets and suggest strongly that gasoline hydrocarbons cause hyaline droplet accumulation by prolonging the half-time of degradation of alpha 2u-globulin.

Leupeptin-mediated alteration of renal phagolysosomes: similarity to hyaline droplet nephropathy of male rats exposed to unleaded gasoline

α 2u-Globulin, a protein of hepatic origin found in the urine of male rats, is accumulated in the kidney cortex during exposure to unleaded gasoline and has been implicated in the development of fuel hydrocarbon-induced nephropathy and renal neoplasia. The principal morphological feature of gasoline-induced nephropathy is accumulation of hyaline droplets (enlarged secondary lysosomes or phagolysosomes) in epithelial cells of the proximal convoluted tubule S1 and S2 segments. Inhibition of cathepsin B (a major lysosomal peptidase) by treatment of male rats with leupeptin causes rapid accumulation of phagolysosomes and α 2u-globulin in the kidney very similar to gasoline exposure. Further, the renal cortical subcellular distribution of α 2u-globulin, determined with an electron microscopic immunochemieal method, is almost totally confined to phagolysosomes following administration of either gasoline or leupeptin. These results, taken together, indicate that the mechanism of nephrotoxicity of gasoline involves inhibition of renal phagolysosomal proteolysis.

Induction of light hydrocarbon nephropathy by p-dichlorobenzene.

In order to clarify the etiology of a dose-related increase in the incidence of tubular cell adenocarcinomas of the kidney in male rats, the nephrotoxicity of p-dichlorobenzene (p-DCB) was investigated in a subchronic study. Groups of ten male and ten female Fischer 344 rats were dosed by gavage with 0 (controls), 75, 150, 300 or 600 mg p-DCB/kg/day in corn oil. Half of the animals were sacrificed after 4 weeks and the remainder after 13 weeks. Increased urinary LDH and epithelial cell excretion and exacerbation of hyaline droplet accumulation in the cytoplasm of renal cortical cells were observed in male rats over the entire dose range investigated. Tubular single cell necrosis, dilated tubules with granular cast formation in the outer zone of the medulla, were evident in male rats after 4 and 13 weeks of treatment with doses of 150-600 mg/kg/day. In female rats there was no indication of a nephrotoxic action of p-DCB. The effects on the kidney, both in their morphological characteristics and the fact that they occur exclusively in male animals, correspond to the light hydrocarbon nephropathy observed as a result of short-term treatment with a number of aliphatic and cyclic hydrocarbons. The development of cortical renal tumors seems to be associated with this kind of kidney damage which is unique to male rats. The literature on this subject generally regards these renal effects as not predictive for man.

Six-month chronic subcutaneous toxicity study of cefodizime sodium in rats

Six-month chronic subcutaneous toxicity study of cefodizime sodium (THR-221) in rats was carried out with dose levels of 3000, 1000, 300 and 100 mg/kg/day. The systemic change observed was slightly decreased spontaneous activity, which appeared only in a very few animals. At the injection site of the animals at 1000 and 3000 mg/kg/day, various cutaneous changes (subcutaneous retention of fluid, incrustation, loss of hair and perforation) were observed. The body weight gains of the males at 1000 and 3000 mg/kg/day were depressed from 1 month of administration onward, but the food consumption was not affected in any group. The water intakes at 1000 and 3000 mg/kg/day were increased. Hematological findings were signs of anemia, a slight decrease in red blood cell count or increases in platelet and/or reticulocyte counts in all THR-221 groups. At 3000 mg/kg/day, increases in white blood cell and neutrophil counts and a decrease in lymphocyte count were also observed. Plasma chemistry revealed decreases in total protein amount and, albumin (A) or globulin (G) amounts, and a decrease or increase in A/G ratio in all compound groups. Autopsy revealed dilation of the cecum and hematoma, dark red spots and yellowish brown spots in the subcutaneous tissue at the injection site in all THR-221 groups. Hypertrophy of the spleen was also noted at 300-3000 mg/kg/day. Changes in organ weights were a decrease in liver weight in all compound groups and an increase in spleen weight at 3000 mg/kg/day. Microscopically, the following were observed: brown granules or hyaline droplets in the epithelium of renal tubules; hemorrhage and inflammatory changes in the subcutaneous tissue at the injection site; and an increased number of lymphocytes or granulocytes in the spleen and bone marrow. Urinalysis and ocular and auditory tests showed no changes related to THR-221. From the present results, the toxicologically non-effective doses of THR-221 are considered to be 300 mg/kg/day for male rats and more than 1000 mg/kg/day for female rats.

Hyaline droplet and a 2u-globulin accumulation in the kidneys of male rats after gasoline administration

Hyaline droplet and a 2u-globulin accumulation in the kidneys of male rats after gasoline administration

A 14-week vapor inhalation toxicity study of methyl isobutyl ketone

In a 2-week probe study male and female Fischer-344 rats and B6C3F1 mice were exposed 6 hr/day to 2000, 500, 100, or 0 ppm methyl isobutyl ketone (MIBK). At 2000 ppm there was a slight increase in male rat liver weight (absolute and relative). The only changes observed histologically were increases in regenerative tubular epithelia and hyalin droplets in kidneys of male rats exposed to 2000 or 500 ppm. Exposure levels for a subchronic study were 0, 50, 250, or 1000 ppm methyl isobutyl ketone vapors 6 hr/day, 5 days per week, for 14 weeks. The 14 weeks of exposure had no adverse effect on the clinical health or growth of rats or mice. Male rats and male mice exposed to 1000 ppm MIBK had a slight but statistically significant increase in liver weight and the liver weight/body weight ratio. Liver weight was also increased slightly in male mice exposed to 250 ppm. No gross or microscopic hepatic lesions related to MIBK exposure were observed. Furthermore, the only microscopic change observed was an increase in the incidence and extent of hyalin droplets within proximal tubular cells of the kidneys of male rats exposed to 250 and 1000 ppm of MIBK. The relevance of the male rat kidney tubular effect to humans is not known. In conclusion, other than the male rat kidney effect, exposure of male and female rats and mice to MIBK at levels up to 1000 ppm for 14 weeks was without significant toxicological effect.