(CANCER RESEARCH 50, 5574-5580, September 1. 1990)

Combined Histochemical and Biochemical Analysis of Sex Hormone Dependence ofFerric Nitrilotriacetate-induced Renal Lipid Peroxidation in ddY Mice1

Shinya Toyokuni,2 Shigeru Okada, Shuji Hamazaki, Yukiko Minamiyama, Yoshihiro Yamada, Ping Liang,

Yasutomo Fukunaga, and Osamu MidorikawaDepartment of Pathology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan

ABSTRACT

Ferric nitrilotriacetate (Fe-NTA) induces renal proximal tubular necrosis, a consequence of lipid peroxidation, that finally leads to a highincidence of renal adenocarcinoma in rats and mice. Male animals aremuch more susceptible than female animals to both effects. Moreover,the distribution of the susceptible proximal tubules is different betweenmale and female animals. The present study investigated the effects ofcastration and sex hormones on Fe-NTA-induced renal lipid peroxidation.Male and female ddY mice were either left untreated, castrated, and/ortreated with testosterone or estriol. Histochemical (reactivity to coldSchiffs reagent) and biochemical (thiobarbituric acid-reactive substance)evaluations were performed l h after the i.p. injection of Fe-NTA (5 mgiron/kg).

Testosterone treatment and/or oophorectomy increased the Schiff pos-itivity of the renal cortical proximal tubules and the amount of thiobarbituric acid-reactive substance (testosterone-treated female > intact female, /' < 0.005; castrated female > intact female, P < 0.1; castratedand testosterone-treated female > intact female, /' < 0.005). In contrast,

estriol treatment and/or orchiectomy decreased the Schiff positivity ofthe renal cortical proximal tubules and the amount of thiobarbituric acid-reactive substance (estriol-treated male < intact male, /' < 0.01; castratedmale < intact male, /' < 0.01; castrated and estriol-treated male < intactmale, /' < 0.005). Estradiol treatment produced similar results to estrioltreatment (estradiol-treated male < intact male, P < 0.005). Castrationand/or administration of the opposite sex hormone reversed the sexdifference in the distribution of proximal tubules susceptible to lipidperoxidation. However, the i.v. injection to male mice, 5 min prior to theFe-NTA treatment, of conjugated estrogen that is promptly excreted viathe urine produced no significant effect. Thus, altered metabolic pathwaysrather than the direct scavenging activity of estrogens seem to be involvedin the sex hormone-dependent difference of lipid peroxidation. Genetically determined sex hormone status appears to have influenced theincidence of Fe-NTA-induced renal adenocarcinoma in intact animals.

INTRODUCTION

NTA3 is a synthetic aminopolycarboxylic acid that efficientlyforms water-soluble chelate complexes with several metal cations at a neutral pH and has been used as a substitute forpolyphosphates in detergents for household and hospital use inthe United States and Canada (1, 2). Experimental models ofiron overload (3) and copper toxicosis (4) have been developedby using Fe-NTA and cupric NTA, respectively. We have hitherto reported the occurrence of acute and subacute renal proximal tubular necrosis and a subsequent high incidence of renaladenocarcinoma in rats and mice given repeated i.p. injectionsof Fe-NTA (5-10). In this model male mice are much moresusceptible than female mice to both acute and subacute neph-rotoxicity and the carcinogenetic effect of Fe-NTA (10, 11),

Received 12/5/89; revised 4/5/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by a Grant-in-Aid from the Ministry ofHealth and Welfare, Japan.

' To whom requests for reprints should be addressed.3The abbreviations used are: NTA, nitrilotriacetic acid; Fe-NTA. ferric nitri

lotriacetate: TBARS, thiobarbituric acid-reactive substance: H&E, hematoxylinand eosin.

which causes lipid peroxidation in the renal proximal tubules(11-14). Lipid peroxidation appears to play an essential role incarcinogenesis, because i.p. aluminum-NTA administration,which induces acute and subacute renal proximal tubular necrosis without lipid peroxidation, leads to no renal neoplasms (9,12). We have recently found differences between male andfemale mice in the vulnerable segment and the severity of Fe-NTA-induced renal lipid peroxidation.4 In the present study,

we investigated the effects of castration and sex hormone treatment in a simplified model with single i.p. injections of Fe-NTA.

MATERIALS AND METHODS

Animals, Castration, and Hormone Treatment. Male and female ddYmice (8-9 weeks of age; males, 32-34 g; females, 27-29 g; ShizuokaLaboratory Animal Center, Shizuoka. Japan) were housed in clearplastic cages and fed a basal diet (II: Funabashi, Japan) and soft waterad libitum. A total of 71 male and 51 female mice were used. Ninetyof them were divided into 10 groups of 10 or 5 (groups 1 and 6) animals(Table 1). The animals of groups 4, 5, 9, and 10 were castrated underpentobarbital anesthesia within 1 week after they were purchased andwere kept untreated for at least 4 weeks before the next procedure. Theanimals of groups 3, 5, 8, and 10 were given a s.c. injection of either10 mg/kg testosterone propionate (Testinon; Mochida, Tokyo, Japan)or 1 mg/kg estriol suspension (Estriel; Mochida) twice a week for 2weeks before the lipid peroxidation experiment. Five animals of eachgroup (except groups 1 and 6) were used solely for testosterone andestriol assays. Six male and 6 female mice were used for histológica!evaluation after the injection of Fe-NTA. The other 20 male animalswere used for the evaluation of estradici and i.v. water-soluble conjugated estrogen treatment. Five mg/kg estradici valerate (Pelanin Depot;Mochida) was injected i.m. once a week for 2 weeks. Four mg/kgconjugated estrogen (principally estrone sodium sulfate and equilinsodium sulfate: Romeda; Mochida), which is immediately excreted inthe urine as estrone, estradici, or estriol, was injected i.v. in the tailvein.

Preparation and Injection of Fe-NTA Solution and Histológica! Examination. The Fe-NTA solution was prepared immediately before useby the method of Awai et al. (3), which was modified. Ferric nitrateenneahydrate (Ishi/u. Osaka, Japan) and nitrilotriacetic acid disodiumsalt (Nakarai, Kyoto, Japan) were each dissolved in deionized water(Millipore Japan, Osaka, Japan) to form 80 miviand 160 mM solutions.They were mixed at the volume ratio of 1:2 (molar ratio, 1:4) withmagnetic stirring at room temperature. The pH was adjusted withsodium hydrogen carbonate (Wako, Osaka, Japan) to 7.4. The irondose was 5 mg/kg and it was injected i.p. into 15- to 16-week-old mice7 days (estradiol), 24 h (estriol and testosterone), or 5 min (conjugatedestrogen) after the last hormonal treatment. The animals other thanthose used for the histológica!evaluation were killed by decapitation 1h after the Fe-NTA administration. These experimental designs werebased on the data of our previous experiments (10-13).'' Both kidneys

of each animal were removed immediately. One was snap-frozen inTissue-Tek* optimum cutting temperature compound (Miles Inc., Elk-hart, IN) with dry ice-cooled acetone (Wako, Osaka, Japan) and stored

* S. Okada, Y. Fukunaga, S. Hamazaki, Y. Yamada, and S. Toyokuni. Histochemical and biochemical demonstration of sex differences in ferric nitrilotriace-tate-induced lipid peroxidation in the mouse kidney: differences in anatomicallocalization and severity. Submitted for publication.

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SEX HORMONES AND RENAL LIPID PEROXIDATION

Table 1 Experimental groupsEach group consisted of 10 or 5 (groups 1 and 6) mice. See text for the details

of drug administration and castration.

Male Female

GroupCastration1-'

234 +5 +Estriol

Fe-NTA GroupCastration-

- 6 -- + 7-+

+ 8 -+ 9 +

+ + 10 +Estriol

Fe-NTA;

+

" -, not treated; +, treated.

Table 2 Histochemical detection oflipid peroxidalion in the renal proximaltubules

See text and Figs. 1-11 for the details of drug administration, castration, andthe interpretation of histochemical detection of lipid peroxidation.

Male Female

Group Cortex Outer stripe Group Cortex Outer stripeControl"Fe-NTA ++E Fe-NTA +Cast Fe-NTA +Cast E Fe-NTA --

Control -Fe-NTA

-t- T Fe-NTA +++ Cast Fe-NTA ++ Cast T Fe-NTA +++î+

* Control, untreated control; E, estriol-treated; T, testosterone-treated; Cast,castrated; —,negative staining; +, positive staining; ++, strongly positive staining;+++, intensely positive staining.

at -40°C for the histochemical detection of lipid peroxidation. The

other was chilled in ice-cold physiological saline solution for theTBARS determination. Six male and 6 female mice were killed at either1 or 3 h, and H&E staining after 10% neutral formalin Fixation wasused for the light microscopic observations.

Histochemical Detection of Lipid Peroxidation. Histochemical detection of lipid peroxidation was performed within 3 days after sacrifice,using cold Schiffs reagent, by the method of Pompella et al. (15).

Measurement of TBARS. The tissue TBARS content was determinedimmediately after sacrifice, using the method of Ohkawa et al. (16)after modification. The pelvis of each kidney was excised before ho-mogenization. We added 0.1% 2,6-di-ferf-butyl-p-cresol (Nacalai Tes-que Inc., Kyoto, Japan) to the reaction mixture to prevent additionalchromophore formation during the assay procedure (17). Protein determination was performed by the method of Lowry et al. (18), usingbovine serum albumin (Sigma, St. Louis, MO) as a standard.

Testosterone and Estriol Assays. Blood was obtained under pernobarbital anesthesia by aortic puncture. Serum was separated immediately and stored frozen at -40°Cuntil determination. Serum testosterone and estriol levels were, respectively, assayed by using the '"I-

testosterone radioimmunoassay kit EIKEN (Eikenkagaku, Tokyo, Japan) and the radioimmunoassay kit Total Hi RIA Pack II (AmershamJapan, Tokyo, Japan), with an ARC-950 7-counter (Aloka Co. Ltd.,Tokyo, Japan).

Statistical Analysis. Statistical analyses were performed by the unpaired t test, which was modified for unequal variances when necessary.

RESULTS

Histochemical Detection of Lipid Peroxidation and I (istologi-cai Findings. The histochemical findings are summarized inTable 2. Kidneys from untreated male and female mice (groups1 and 6) were unstained after exposure to cold Schiffs reagent,except for the elastic fibers of arterial walls. One h after the Fe-NTA injection, male mice (group 2) showed patchy Schiffpositivity in the renal proximal tubules (Fig. 1), at which timeroutine histológica! examination with H&E staining showed noremarkable changes. At 3 h, however, patchy degeneration ofthe cortical proximal tubular epithelium could be observed,with pyknotic nuclei and necrotic debris in the tubules (Fig. 2).

In contrast, female mice 1 h after the Fe-NTA injection(group 7) showed weaker and limited Schiff positivity of the

Fig. 1. Male mouse treated with Fe-NTA (5 mg iron/kg) 1 h before sacrifice.Patchy distribution of Schiff positivity in the cortical proximal tubules, x 40.

Fig. 2. Male mouse 3 h after Fe-NTA injection. Palchy degeneration of theproximal tubular epithelium with pyknotic nuclei (small arrowheads). Necroticdebris in the tubules (large arrowheads). H&E. x 200.

outer stripe of the outer medulla, which also consisted ofproximal tubules (19), and no Schiff positivity in the corticalproximal tubules (Fig. 3). H&E-stained sections showed nosignificant changes either at 1 or 3 h (Fig. 4).

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Fig. 3. Female mouse l h after Fe-NTA injection. Diffuse patchy Schiff- Fig. 5. Estriol-treated male mouse l h after Fe-NTA injection. Weak patchypositive areas in the outer stripe of the outer medulla (asterisk), x 40. Schiff positivity in the cortical proximal tubules and faint Schiff positivity of the

outer stripe of the outer medulla (asterisk), x 40.

•

Fig. 4. Female mouse 3 h after Fe-NTA injection. No significant changes inthe cortex. H&E. x 200.

positivity of the renal cortical proximal tubules but increasedthe Schiff positivity of the outer stripe of the outer medulla(groups 3-5) (Figs. 5-7). Estradici treatment revealed the sameeffect as estriol treatment (data not shown). In contrast, testosterone treatment and/or oophorectomy increased the Schiffpositivity of the renal cortical proximal tubules but decreased

Fig. 6. Castrated male mouse l h after Fe-NTA injection. Dispersely distributed Schiff positivity in the cortical proximal tubules and faint Schiff positivityof the outer stripe of the outer medulla (asterisk), x 40.

the Schiff positivity of the outer stripe of the outer medulla(groups 8-10) (Figs. 8-11). Schiff-positive material appearedin the proximal tubules just after the glomerular filtration, andglomeruli and some of the cortical proximal tubules were com-

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r -

Fig. 7. Castrated and estriol-treated male mouse l h after Fe-NTA injection.No Schiff positivity in the cortical proximal tubules and faint Schiff positivity ofthe outer stripe of the outer medulla (asterisk), x 40.

Fig. 9. Castrated female mouse l h after Fe-NTA injection. Dispersely distributed Schiff-positive areas in the cortical proximal tubules and faint Schiffpositivity of the outer stripe of the outer medulla (asterisk), x 40.

*»<

Fig. 8. Testosterone-treated female mouse l h after Fe-NTA injection. Strongpatchy Schiff positivity of the cortical proximal tubules, x 40.

pletely spared in either group (Fig. 11).Measurement of TBA-reactive Substance. Testosterone treat

ment and/or oophorectomy increased the TBARS content (testosterone-treated female > intact female, P < 0.005; castratedfemale > intact female, P < 0.1; castrated and testosterone-

Fig. 10. Castrated and testosterone-treated female mouse I h after Fe-NTAinjection. Very strong patchy Schiff positivity in the cortical proximal tubules.X40.

treated female > intact female, P < 0.005). In contrast, estrioltreatment and/or orchiectomy decreased the TBARS content(estriol-treated male < intact male, P < 0.01; castrated male <intact male, P < 0.01; castrated and estriol-treated male <

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v;. •••»••*/

-r* j* ' » A

Fig. 11. Higher magnification of Fig. 10. Schiff-positive material appears inthe proximal tubules just after the glomerular filtration. Glomeruli and someproximal tubules are completely spared, as seen in the other groups, x 400.

intact male, P < 0.005) (Table 3. Fig. 12). Male mice treatedwith estradici (58.5 ±9.5 nmol malondialdehyde/100 mg protein; mean ±SE; P < 0.005 versus intact male; n = 5) showedsimilar results as those treated with estriol. Intravenous pretreatment with water-soluble conjugated estrogen (134.9 ±22.6, n = 5) produced no significant change in the amount ofTBARS in comparison with animals without pretreatment(126.4 ±23.0 nmol malondialdehyde/100 mg protein; mean ±SE; n = 5).

Assay of Testosterone and Estriol. Serum levels of each sexhormone in animals of groups 2-5 and 7-10 at sacrifice areshown in Table 4.

DISCUSSION

Castration and/or treatment with the opposite sex hormonereversed the sex differences in the distribution and severity oflipid peroxidation in the renal proximal tubules in this murinemodel.

It is known that androgen and estrogen receptors exist in therenal proximal tubules (20-22). It has been reported that theexpression of several genes (23, 24) and the incorporation ofuridine into RNA (25) are regulated by androgens in the murinekidney. The differences between male and female mice in thedistribution and activity of several enzymes (26. 27) as well asmicroscopic morphological differences (26-30) have also beenreported in relation to sex hormones.

It was of note that not only estradiol but also estriol had adramatic inhibitory effect on lipid peroxidation in this model.This is consistent with the recent finding that pregnancy, duringwhich estriol shows the most prominent increase among estrogens (31), lowers renal and hepatic lipid peroxidation (32, 33).It has also been reported that estrogen itself (estrone, estradiol,

and estriol), but not testosterone, is an antioxidant in vitro (34-36). We believe, however, that a role for estrogen itself as anantioxidant is unlikely in this model for the following reasons.Firstly, there was a change in the distribution of Fe-NTA-induced-lipid peroxidation of the renal proximal tubules between male and female mice (Figs. 1 and 3). Secondly, testosterone markedly promotes lipid peroxidation, as shown in Figs.8, 10, and 12. Thirdly, an increase in the estrogen content ofthe renal tubular lumens by i.v. estrogen administration had nosignificant effect on the TBARS content. Fourthly, the estrogen-rich environment attained in vitro (34-36) seems unlikelyto be obtained in the luminal membrane of the renal proximaltubules in vivo when the total amount of estrogen administeredis considered. Finally, in another model of kidney damageassociated with lipid peroxidation produced by phenylbutazone,female rats are more susceptible than male animals (37).

We have recently reported, in in vitro experiments, that thereduction from ferric to ferrous state and the immediate furtherautoxidation was essential for the induction of oxygen radicalsby Fe-NTA, and we suggested that lipid peroxidation wasinitiated at the luminal membrane of the proximal tubules inthis model (8, 38, 39). This was confirmed by our present studyin which Schiff-positive material appeared in the proximaltubules just after the glomerular filtration (Fig. 11). We thusspeculate that either functional heterogeneity in the ability toreduce iron in the proximal tubular lumen, the distribution ofantioxidants such as vitamin E (40, 41). Superoxide dismutase,glutathione (42, 43), and metallothionein (44), or the renaltubular localization of unsaturated lipids that provide the majorsubstrates for lipid peroxidation is closely associated with thesex hormones.

We demonstrated by modifications of sex hormones that thesusceptibility of renal proximal tubules to lipid peroxidation bythis iron chelate was genetically determined. The data fromboth biochemical quantitative detection and histochemical localization supported this conclusion. These findings seemstrongly connected to the difference in carcinogenesis betweenmale and female animals (10). An experiment assessing theeffects of castration on Fe-NTA-induced renal carcinogenesisis now in progress.

In this model TBARS reaches the maximum value from 30min to 2 h after the administration, followed by cortical proximal tubular necrosis at no later than 3 h and regenerationthereafter.4 In the case of repeated administration, morpholog

ically atypical regenerative cells with positive iron stainingappear as early as at 10 days, and atypical cells and atypia itselfincrease according to the experimental days (5-7, 9, 10). Wealso observed the process from dysplastic cells to carcinoma, asis seen in estrogen-induced renal adenocarcinoma of hamsters(45). The exact significance of lipid peroxidation, whether it issolely a concomitant of oxygen radical-induced DNA damagethat may lead to carcinogenesis (46) or whether its productsincluding alkenals and malonaldehydes ( 15,47,48) play a major

Table 3 Renal TBARS I h after ¡hei.p. injection of ferric nitrilotriacetate (5 mg ¡ron/kg)The abbreviations are shown in the legend to Table 2. Values are mean ±SE (n = 5).

Group

Male Female

TBARS (nmol M DA/100 mgprotein) Group

TBARS (nmol MDA/100 mgprotein)

ControlFe-NTAE

Fe-NTACastFe-NTACast

E Fe-NTA20.8119.566.563.450.30.824.7°i.r5.2C2.2»ControlFe-NTAT

Fe-NTACastFe-NTACast

T Fe-NTA20.0

±0.233.4±2.4°''1I6.0±

10.8*40.2+4.3''148.8±13.5*

a-e* refer to the legend of Fig. 12.

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Male Mice Female Mice

Fig. 12. Renal thiobarbituric acid-reactive substances l h after the i.p. injectionof ferric nitrilotriacetate (5 mg iron/kg). See text for the details of castration andsex hormone administration. Control, untreated control; Fe, Fe-NTA-treated: E.estriol-treated; T, testosterone-treated; Cast, castrated; a. P < 0.005 versus control;b, P < 0.005 versus Fe; c. P < 0.01 versus Fe; d, P < 0.1 versus Fe; e, P < 0.005versus Fe/male mice. Except for e, comparison was performed between groups ofthe same sex (mean ±SE, n = 5).

Table 4 Serum hormone levels of castrated and/or hormone-treated mice

See Table 1 for the experimental groups. Testosterone propionate (10 mg/kg)or estriol suspension (1 mg/kg) was administered s.c. twice a week for 2 weeks.Serum was obtained at the sacrifice of animals that were given i.p. injections offerric nitrilotriacetate (5 mg iron/kg) 24 h after the last hormonal treatment.Values are mean ±SE (n = 5).

Group12345678910Testosterone(ng/ml)ND"<IND<1N

DND<117.2

±3.2<111.7±

2.0Estriol

(ng/ml)ND<28.0

±3.0<23.6

±1.3ND<2ND<2ND

" ND. not determined.

role in mutagenicity (47) or initiation (49), requires furtherstudy to be elucidated.

The histochemical detection of lipid peroxidation is helpfulin interpreting the significance of borderline data obtained bybiochemical studies, like that for the castrated female mice(Figs. 3, 9, and 12). The precise mechanism of sex hormonefunction in carcinogenesis in this model should be furtherinvestigated, since the carcinogenetic process is completed in arelatively short period of time (10) with male preponderancethat is similar to human renal cell carcinoma (50).

ACKNOWLEDGMENTS

The authors thank Dr. Yoshiro Niitsu (Department of InternalMedicine, Sapporo Medical College, Sapporo, Japan) for advice andencouragement and Yoshinobu Toda, Hitoshi Abiru, Makio Fujioka,Shinji Toh, and Hisae Yorisawa for the technical assistance.

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ANNOUNCEMENTS

GENOMIC INSTABILITY AND CANCER ChairpersonsFebruary 21—26,1994 Harold L. Moses, Nashville, TNBanif Springs Hotel, Bernd Groner, Basel, Switzerland

Banif, Alberta, Canada

Chairpersons

Thea D. Tlsty, Chapel Hill, NC RECENT DEATHSLawrence A. Loeb, Seattle, WA

We regret to report the deaths ofDr. Zalmen A. Arlin ofthe New YorkMedical College, Valhalla, NY; Dr. M. Adrian Gross of Silver Spring,MD; Dr. Arthur Worth Ham of Markham, Ontario, Canada Dr. S.

GROWTH FACTORS, DEVELOPMENT, Charles Kasdon of Boston, MA; and Dr. Einar Pedersen of The NorAND CANCER wegian Radium Hospital, Oslo, Norway. Drs. Arlin and Gross were

(Joint Meeting with the Friedrich Miescher-Institut) Active Members, Dr. Pedersen was a Corresponding Member, and Drs.March 5—11, 1994 Ham and Kasdon were Emeritus Members ofthe American Association

Congress Center, Interlaken, Switzerland for Cancer Research.

CALENDAR OF EVENTS

Eighteenth Annual Myron Karon Memorial Lectureship—HumanGene Therapy, delivered by Dr. W. French Anderson, November 20, 1992,Childrens Hospital ofLos Angeles Santa Anita Lecture Hall, Los Angeles, CA. Credits: 1 hour CME Category I. Contact: Edward Kim, Children's

Hospital of Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027. Telephone: (213) 669-5653.

Arizona Cancer Center Fifth International Workshop on Chromosomes in Solid Tumors, January 10—12,1993, Doubletree Inn, Tucson, AZ.Contact: Nancy Rzewuski, Arizona Cancer Center, 1515 North Campbell Avenue, Tucson, AZ 85724. Telephone: (602) 626-6044; FAX: (602)626-2284.

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Seventh International Conference on the Adjuvant Therapy of Cancer, March 10—13,1993, Tucson Convention Center, Tucson, AZ. Abstractdeadline: December 1, 1992. Contact: Nancy Rzewuski, Arizona Cancer Center, University of Arizona College of Medicine, 1515 N. CampbellAvenue, Room 2933, Tucson, AZ 85724. Telephone: (602) 626-2276; FAX: (602) 626-2284.

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Second Consensus Development Conference on the Treatment of Radiation Injuries: Marrow Aplasia, Sepsis, Gastrointestinal Injury, andRadiation Accidents, April 14—17,1993, Bethesda, MD. Contact: Dr. Doris Browne, Armed Forces Radiobiology Research Institute/MRA, 8901Wisconsin Avenue, Bethesda, MD 20889-5603. Telephone: (301) 295-0316; FAX: (301) 295-5673.

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International Conference on Engineered Cancer Vaccines, September 29—October2, 1993, Fairmont Hotel, San Francisco, CA. Abstract deadline:March 1, 1993. Contact: Cass Jones, Vaccines Conferences, 7916 Convoy Court, San Diego, CA 92111. Telephone: (619) 565-9921; FAX: (619)565-9954.

Third International Congress on Oral Cancer, January 22—25,1994, Madras, Tamil Nadu, India. Contact: Arati Walia, Organizing Secretary, D-l,Kalindi Colony, New Delhi I 10065, India. Telephone: 91-01 1-684-9399; FAX: 91-011-684-8343.

Errata

Two errors have been found in the article by Toyokuni et aL, entitled “CombinedHistochemical and Biochemical Analysis of Sex HormoneDependence of Ferric Nitrilotriacetate-induced Renal Lipid Peroxidation in ddY Mice,―which appeared in the September 1, 1990 issue of CancerResearch (pp. 5574—5580).In Table 1, the column labeled “Estriol―under the “Female―heading should have been labeled “Testosterone.―Inaddition, in the legend to Fig. 3, the word “patchy―should been omitted from the expression “diffusepatchy Schiff-positive areas.―

Coauthor Timothy J. Guzi's name was misspelled as Timothy J. Cuzi in the article by Leteurtre et a!., titled “RationalDesign and MolecularEffects of a New Topoisomerase II Inhibitor, Azatoxin,―which appeared in the August 15, 1992 issue of Cancer Research (pp. 4478—4483).

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1990;50:5574-5580. Cancer Res   Shinya Toyokuni, Shigeru Okada, Shuji Hamazaki, et al.   Lipid Peroxidation in ddY MiceHormone Dependence of Ferric Nitrilotriacetate-induced Renal Combined Histochemical and Biochemical Analysis of Sex

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