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 DOI: 10.1177/019262339702500209

1997 25: 195Toxicol PatholSoochong Kim, Charles W. Qualls, jr, Gunda Reddy and Eric L. Stair

1,3,5-Trinitrobenzene-Induced Alpha-2u-Globulin Nephropathy  

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1,3,5-Trinitrobenzene-InducedAlpha-2u-Globulin Nephropathy*1

SOOCHONG KIM,2 CHARLES W. QUALLS, JR.,2GUNDA REDDY,3 AND ERIC L. STAIR2

2Department of Veterinary Pathology, College of Veterinary Medicine,Oklahoma State University, Stillwater, Oklahoma 74078, and3U.S. Army Center for Health Promotion and Preventive Medicine,

Aberdeen Proving Ground, Maryland 21010

* Address correspondence to: Dr. Charles W. Qualls, Jr., Departmentof Veterinary Pathology, College of Veterinary Medicine, OklahomaState University, Stillwater, Oklahoma 74078.

1 The views, opinions, and/or findings contained in this report arethose of the authors and should not be construed as official Departmentof the Army position, policy, or decisions, unless so designated by otherofficial documentation. Research was conducted in compliance with theAnimal Welfare Act and other federal statutes and regulations relatingto animals and experiments involving animals and adheres to principlesstated in the Guide for the Care and Use of Laboratory Animals, NIHPublication No. 86-23, 1985 edition.

ABSTRACT

Male and female Fischer-344 (F-344) and male NCI-Black-Reiter (NBR) rats were dosed with 0, 35.5, or 71 mg 1,3,5-trinitrobenzene(TNB)/kg/day for 10 days. Male F-344 rats were dosed with TNB (0 and 35.5 mg/kg) for 20 and 30 days. Hematoxylin and eosinand Mallory-Heidenhain stains and alpha-2u-globulin and proliferating cell nuclear antigen immunohistochemical stains were per-formed on kidney sections. All treated male F-344 rats exhibited dose-related accumulation of hyaline droplets containing alpha-2u-globulin in proximal tubules. The kidney weights were significantly increased in male and female rats treated with TNB. Significantincreases in cell proliferation in proximal tubules were observed in male F-344 rats. Renal changes observed in TNB-treated ratsappeared identical to those from other chemicals that induce alpha-2u-globulin nephropathy in male rats. No hyaline droplet accu-mulation was found in female F-344 and male NBR rats at any doses. We can conclude that TNB induces dose-related exacerbation

of hyaline droplets containing alpha-2u-globulin in male rat kidney and subsequent cell proliferation.

Keywords. Nitroaromatic compound; munition chemical; hydrocarbon nephropathy; hyaline droplet; immunohistochemistry; pro-liferating cell nuclear antigen (PCNA); cell proliferation; Fischer-344 rat

INTRODUCTION

The nitroaromatic compound 1,3,5-trinitrobenzene(TNB), a process and environmental byproduct of 2,4,6-trinitrotoluene, has been detected as an environmentalpollutant of surface water, ground water, and soil nearmunition plants and production waste disposal sites (53,55). Recently, it has been reported that TNB exposurecan induce renal toxicity characterized by hyaline dropletaccumulation in proximal tubules in male rats (37-39).

Alpha-2u-globulin (alpha-2u-G) is a primary constit-uent of hyaline droplets in male rats and the essentialfactor that renders male rats susceptible to chemicallyinduced hyaline droplet nephropathy (4, 22, 31). Hyalinedroplet nephropathy is manifested as protein droplets inproximal tubules and is caused by the reversible and non-covalent binding of a variety of chemicals or their me-tabolites to alpha-2u-G (6, 8, 20-26, 30, 44-47, 49, 50).The formation of alpha-2u-G-chemical complex resultsin decreased hydrolysis in lysosomes of the proximalconvoluted tubules, subsequent lysosomal overload, anddegeneration and necrosis of the proximal tubular epithe-lial cells followed by compensatory renal tubular cell

proliferation (44, 46, 50, 51, 58). It is believed that asustained increase in cell proliferation leads to the de-velopment of renal tubular neoplasms (9, 43, 44). Thisrenal disease occurs in male rats but not in female ratsor either sex of mice, guinea pigs, hamsters, dogs, ormonkeys (1, 28, 33, 46, 50, 51, 57). The only male ratstrain known to not develop this renal disease is the NCI-Black-Reiter (NBR) rat (9, 10, 23, 40). Chatterjee et al(7) reported that the male NBR rat does not synthesizealpha-2u-G in the liver.

Because reports of the renal toxicity of TNB are con-fined to a few abstracts, the purpose of the current studywas (a) to determine the role of alpha-2u-G in the patho-genesis of the hyaline droplet nephropathy caused byTNB by comparing the renal lesions between male F-344rats and female F-344 and male NBR rats, (b) to identifythe nature of the protein droplets by immunohistochem-istry, and (c) to evaluate the renal cell replication by im-munohistochemistry using proliferating cell nuclear an-tigen (PCNA), an endogenous cell replication marker.

MATERIALS AND METHODS

TNB (99.83% purity) was obtained from the NavalSurface Center (Silver Spring, MD) and the purity of thecompound was confirmed by high-performance liquidchromatography. TNB was mixed with corn oil in a Pot-ter-Elvehjem grinder daily prior to dosing. The oral LD,ofor TNB in rats has been determined to be 284 mg/kgfor combined sexes (13). The one-eighth (35.5 mg/kg)and one-fourth (71 mg/kg) LD,, doses were selected forthese studies based on findings from a previous study(37). Twenty-six male and 12 female Fischer (F-344) rats

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and 12 male NBR rats aged between 9 and 11 wk wereused in the experiment. The animals were housed singlyand provided feed (Purina Rat Chow) and tap water adlibitum. After a 2-wk acclimatization (12-h light/12-hdark cycle, 72 ±- 2°F, 50 ± 10% relative humidity), maleNBR and female F-344 rats were exposed to TNB for 10days. Male F-344 rats were stratified by body weight andrandomly assigned to 3 exposure periods of 10, 20, and30 days. The rats were further randomized to dose groupwithin each exposure period. Within 10-day exposure pe-riods, 4 rats were separated into 3 TNB dose groups of0, 35.5, and 71 mg/kg. Within 20- and 30-day exposureperiods, 3 rats served as controls and 4 rats were admin-istered TNB at 35.5 mg/kg. The rats were gavaged withTNB in corn oil by feeding needle. Controls received thesame volume of corn oil. All treatments were conductedonce a day between 9:00 am and 10:00 am for 10, 20,and 30 days and body weights were recorded daily. Allrats were fasted for 15 hr prior to termination. At the endof each experimental period, all rats were anesthetizedwith Metofane© (Pitman-Moore, Mundelein, IL). For re-nal morphology, the kidneys were fixed by retrogradeperfusion via the abdominal aorta with sodium phosphatebuffer (pH 7.4) containing 2% paraformaldehyde and 1 %glutaraldehyde, as previously described (45). The left

kidney was further fixed in 10% neutral-buffered for-malin and embedded in paraffin. The liver, kidney,spleen, testicle, and brain were weighed at necropsy. Sec-tions of all parenchymal organs were collected in 10%neutral-buffered formalin, embedded in paraffin, sec-

tioned at 5-6 )JLm, and stained with hematoxylin and eo-sin. Sections of kidney were stained with Mallory-Hei-denhain stain (5) for hyaline droplets. Immuno-histochemical staining with anti-alpha-2u-G antiserum(courtesy of Dr. S. J. Borghoff, Chemical Industry Insti-tute of Toxicology, Research Triangle Park, NC) was per-formed using an avidin-biotin complex on kidney sec-tions as described by Burnett et al (4) and Kurata et al(23). Immunohistochemical staining for PCNA was doneon kidney sections from all male F-344 rats. The stainingprocedure was modified from several sources (14, 16, 17,40). Microwave pretreatment of tissue section in an an-tigen retrieval solution was performed. The percentage ofS-phase cells was determined by examining 35,000-45,000 cells in P, segments of proximal tubules per slide,2 slides per animal, using light microscopy. At least 20random fields of each kidney section were used, andcounts were completed manually using an ocular grid ret-icle.

Statistical differences were determined by usingPC-SAS (SAS Institute Inc., Cary, NC). All data weretested for homogeneity of variances prior to analysis ofvariances (Proc. GLM, SAS), and heterogeneity of vari-ances was not observed. When significant F-values wereobtained, the method of least square means (LSM) wasused to determine significant differences between treat-ment means. Unless otherwise mentioned, p-values of~0.05 were considered to be significant.

RESULTS

The kidney/body weight ratios showed dose-related in-creases in all treated rats with TNB for 10 days (Table

TABLE I.-Kidney weights of rats dosed to TNB.

a mean ± SD.

b Significantly different from control within the same exposure period at p <

0.05.1 Significantly different from control within the same exposure period at p <

0.01.

I), and the weight changes were of similar magnitude inmale and female F-344 rats. The kidney weights weresignificantly (p < 0.01) increased in both groups of treat-ed male F-344 rats compared to controls. NBR rats re-ceiving 71 mg/kg of TNB and all treated female F-344rats showed significant (p < 0.05) increases in kidneyweights, whereas kidney weights were not statisticallydifferent between controls and treated (35.5 mg/kg) NBRrats. There were no statistically different changes in kid-ney weights between male F-344 rats receiving 0 or 35.5mg/kg for 20 days. (There was 1 control rat that hadincreased kidney weights compared to the other controlrats. If this rat was regarded as an outlier, there would besignificant changes in rats dosed for 20 days.) The rela-tive kidney weights of male F-344 rats receiving a doseof 35.5 mg/kg for 30 days were significantly (p < 0.05)higher than controls. When changes in kidney weightswere compared after 10 and 20 days of dosing, there wasno apparent time-dependent relationship in male F-344rats treated with TNB.

Dose-related accumulation of hyaline droplets was ob-served in the H&E (Fig. 1) and Mallory-Heidenhainstains (Fig. 2) of male F-344 rats treated with TNB incomparison to female F-344 and male NBR rats. Thesechanges were detected only in the renal proximal con-voluted tubular epithelial cells. By immunohistochemis-try, prominent increases in alpha-2u-G staining area andintensity were detected in hyaline droplet accumulatingregions of the kidney sections of TNB-treated male F-344rats (Fig. 3). Alpha-2u-G was not detected in the renalsections from any of the male NBR and female F-344rats (Fig. 4), regardless of treatment of TNB. Pathologicalchanges were characterized by prominent accumulationof protein droplets, which increased in size and number,dilated tubules with tubular protein, exfoliation of epi-thelial cells, and various stages of tubular necrosis. Theprotein droplets were pleomorphic, large, and globular topolyangular in shape. Control male F-344 rats showed asmall numbers of hyaline droplets, which were usuallysmall and round (Fig. 5).When comparing the dose-related changes in male

F-344 rats among 10-, 20-, and 30-day treatment groups,

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... - - -.

FIG. 1.-Renal cortex from a male F-344 rat dosed to 35.5 mg/kg ofTNB for 30 days. Note the accumulation of hyaline droplets, exfoliationof renal tubular epithelial cell (arrow), and necrotic tubular epithelialcell (arrowhead). H&E. x 370.

the protein droplet accumulation and alpha-2u-G concen-tration increased with longer exposure period. No otherdifferences between the kidneys of control male F-344rats and control or treated female F-344 or male NBRrats and those of treated male F-344 rats were demon-strated by light microscopy.PCNA immunostaining revealed S-phase cells char-

acterized by dense nuclear immunoreactivity (Fig. 6).Only S-phase cells in the P2 segment of renal proximaltubules were counted, and the percentage of cells in Sphase of each group is summarized in Table II. MaleF-344 rats receiving 35.5 mg/kg (p < 0.001) or 71 mg/kg(p < 0.05) showed significant increases in S-phase cellsand an inverse dose relationship at 10 days. Rats receiv-

FIG. 2.-Mallory Heidenhain staining of renal cortex from a maleF-344 rat dosed to 35.5 mg/kg of TNB for 10 days. Note the exacer-bated globular to polyangular droplet accumulation in proximal con-voluted tubules. x 145.

.._- ~,

FIG. 3.-Immunohistochemical staining of alpha-2u-G from a maleF-344 rat dosed to 35.5 mg/kg of TNB for 30 days. Note increase inthe quantity and the number of droplets in proximal covoluted tubulescompared to the control in Fig. 5. x 35.

ing 35.5 mg/kg for 20 days showed slightly decreasedcell proliferation compared to controls, which was notstatistically significant. A greater magnitude (p < 0.001)of increased cell proliferation was observed in rats re-ceiving 35.5 mg/kg for 30 days (Fig. 7).

DISCUSSION

Renal toxicity has been reported with the nitroaromaticcompound nitrotoluene in male rats (12). An increasedaccumulation of hyaline droplets within the cytoplasmand lumen of the renal tubules associated with an in-creased renal concentration of alpha-2u-G was observed.The results from the present study demonstrated that ex-posure to TNB in male rats produced dose-related in-creases in hyaline droplet formation in the renal proximal

FIG. 4.-Immunohistochemical staining of alpha-2u-G of kidney froma male NBR rat dosed to 71 mg/kg of TNB for 10 days. Note thecomplete absence of alpha-2u-G-positive droplets. X390.

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FIG. 5.-Immunohistochemical staining of alpha-2u-G from a controlmale F-344 rat. A small number of droplets are present in proximalconvoluted tubules. X35.

convoluted tubular epithelium. There were dilated tubuleswith intratubular protein and necrosis of tubular cells.These observations have been reported following treat-ment with chemicals that cause hyaline droplet nephrop-athy (6, 8, 20-26, 30, 44-47, 49, 50, 52, 56).The liver of male NBR rats and all strains of female

rats do not synthesize alpha-2u-G (7). Hyaline dropletswere not detected in female F-344 and male NBR rats at

any doses, and these results are comparable to thosefound in other studies of alpha-2u-G inducers (1, 46, 50,57). The lack of alpha-2u-G and hyaline droplets in kid-neys of female F-344 and male NBR rats suggests thepivotal role of alpha-2u-G in the pathogenesis of hyalinedroplet nephropathy induced by TNB.

Borghoff et al (3) reported that kidney/body weightratios were not changed in rats exposed to hyaline drop-

FIG. 6.-Immunohistochemical staining of renal cortex from a controlmale F-344 rat. Several PCNA-positive nuclei (arrow) are present.X 155.

TABLE II.-Cell proliferation in renal proximal tubules of male F-34,rats dosed to TNB.

a Mean ± SD.b Significantly different from control within the same exposure period at p <

0.001.~ Significantly different from control within the same exposure period at p <

0.05.

let-inducing agents, such as unleaded gasoline and 2,2,4-trimethylpentane. However, dose-related increases in kid-ney/body weight ratios were observed in all rats treatedwith TNB. These results are comparable to those reportedfollowing exposure to decalin, D-limonene, and TNB inmale F-344 rats (21, 36). Kurata et al (23) observed dose-related increases in kidney/body weight ratios in malerats, but not in female rats, exposed to sodium barbitaland diethylacetylurea. The mechanism of increased kid-ney/body weight ratio is unclear, but it is possibly due tothe metabolism of TNB. It appears that it is not solelyrelated to alpha-2u-G formation because similar changeswere seen in female F-344 and male NBR rats that didnot develop alpha-2u-G nephropathy.A variety of chemicals bind reversibly to alpha-2u-G,

which appears to reduce the hydrolysis of the protein inproximal tubular epithelial lysosomes. Viau et al (54)demonstrated that alpha-2u-G-inducing chemicals do notaffect the rate of hepatic synthesis of alpha-2u-G and pro-tein resorptive capacity of renal proximal tubular cells.This study supports the hypothesis that decreased hydro-

FIG. 7.-Immunohistochemical staining of renal cortex from a maliF-344 rat dosed to 35.5 mg/kg of TNB for 30 days. Numerous PCNApositive nuclei with several severely altered proximal tubules (arroware present (note increased numbers compared to the control in Fig. 6)X 155.

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lysis of alpha-2u-G-chemical complex is the major factoraffecting renal accumulation of protein droplets. The in-creased resistance to hydrolysis could result in exacer-bation of hyaline droplet accumulation in P2 segments ofthe proximal tubules and cytotoxicity followed by com-pensatory proliferative responses in proximal tubules (44,46, 50, 51, 58). It has been assumed that sustained pro-liferation of renal tubule cells is associated with incidenceof renal tubular neoplasms in alpha-2u-G nephropathy (9,43, 44). It is believed that at least 6 mo of exposure tohyaline droplet-inducing agents, such as unleaded gaso-line or D-limonene, with resulting sustained cell replica-tion leads to the development of renal tubular neoplasms(43). Significant increases in proliferating cells (S phase)in P2 segments of proximal tubules were observed in ratstreated with TNB for 30 days, and these results werecomparable to those reported following exposure to hy-aline droplet-inducing chemicals, such as unleaded gas-oline, sodium barbital, and diethylacetylurea (23, 27).TNB-treated male rats showed 3.5-fold increases, where-as others exposed to unleaded gasoline for 18 days haveshown 5-8-fold increases and those exposed to sodiumbarbital for 10 wk have shown 3-6-fold increases. TNB-treated male rats demonstrated 2-fold increases after 10

days, whereas others receiving perchloroethylene andpentachloroethane have shown 2-3.5-fold increases (15).This study also revealed the changes in cell proliferationamong 10-, 20-, or 30-day dose groups. These time-de-pendent changes in cell proliferation have not been re-ported in any other studies, and the mechanism related tothis result is not fully understood at this point. Althoughchronic renal toxicity evaluation of TNB was not per-formed in this study, it is hypothesized that TNB wouldcontribute to the promotion of renal tumors in alpha-2u-Gnephropathy based on other investigators’ studies withother alpha-2u-G-inducing chemicals (10, 47). However,it has been reported that 1-(aminomethyl)cyclohexaneaceticacid, an alpha-2u-G-inducing agent, does not induce renalcancer in male rats treated for 2 yr (11). This may berelated to the insufficient renal tubular injury to effec-tively promote tumor formation. We have recentlylearned that rats fed TNB had testicular damage and norenal tumors. These rats were on a diet containing 5, 60,and 300 ppm of TNB (calculated 0.22, 2.64, and 13.44mg TNB/kg/day) for 2 yr (Dr. T. V. Reddy, personal com-munication). It is likely that the failure to produce renaltumors is due to the toxic effects of TNB on testicleswith resulting decrease in testosterone levels. It has beenknown that the decreased testosterone levels results in thedecreased synthesis of alpha-2u-G (41). The lower doseof TNB in these studies is also a likely contributing fac-tor.

It was necessary to identify the nature of accumulatingproteins in rat kidneys because abnormal accumulation ofrenal hyaline droplets that is not associated with alpha-2u-G has been observed in several disease processes. Therenal injury including hyaline droplet accumulation sec-ondary to histiocytic sarcoma in male and female rats andto multiple myeloma in humans have been identified aslysozyme and light-chain immunoglobulins, respectively(18, 34). Excess amounts of lysozyme have been detected

in the nephropathy of monocytic and myelomonocyticleukemia in humans (29, 35). Immunohistochemistrydemonstrated the increase in renal alpha-2u-G accumu-lation in treated male rats. This indicates that alpha-2u-Gis a primary constituent of the hyaline droplets inducedby TNB.

It has been known that several proteins isolated fromserum and urine of various species including humansshare extensive amino acid sequence homology with al-pha-2u-G (32, 48). It is unlikely that humans develophyaline droplet nephropathy and renal cancer followingtreatment with alpha-2u-G-inducing agents (2, 19). Thus,we can assume that despite the presence of TNB as anenvironmental contaminant humans are unlikely to de-velop hyaline droplet nephropathy induced by TNB.

In conclusion, the current study confirms that alpha-2u-G is important for hyaline droplet accumulation inmale F-344 rats after administration of TNB. TNB in-duced dose- and time-dependent exacerbation of hyalinedroplets and subsequent renal tubular cell proliferation.The cause of the increased kidney/body weight ratio thatwas seen in all groups exposed to TNB even in the ab-sence of alpha-2u-G needs to be determined.

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