(CANCER RESEARCH 43, 5662-5667, December 1983]

Chromium(VI)-induced DMA Lesions and Chromium Distribution in RatKidney, Liver, and Lung1

Michael J. Tsapakos, Thomas H. Hampton, and Karen E. Wetterhahn2

Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755

ABSTRACT

DNA lesions were detected in rat organ nuclei following an i.p.injection of sodium dichromate. Kidney, liver, and lung nucleiwere examined for DNA interstrand cross-links, strand breaks,and DNA-protein cross-links using the alkaline elution technique.The time course for formation of cross-links in kidney nucleirevealed the presence of DNA interstrand and DNA-proteincross-links 1 hr after injection of sodium dichromate. By 40 hr inkidney, DNA interstrand cross-links had been repaired, but DNA-protein cross-links persisted. In liver nuclei, the time course forformation of cross-links after injection of dichromate showed amaximum in DNA-protein cross-linking at 4 hr and a maximumin DNA interstrand cross-linking at 2 hr. By 36 hr, in the liver,

both types of lesions had been repaired. In lung nuclei, bothDNA interstrand and DNA-protein cross-links were observed 1hr after dichromate injection; however, by 36 hr, only DNA-protein cross-links persisted. No DNA lesions were detectable in

kidney 1 hr after an i.p. injection of chromium(lll) chloride. Chromium distribution in rat kidney, liver, and lung was measuredand is discussed with respect to the observed DNA lesions. Thelung and kidney may be more sensitive than liver to chromium-

induced DNA damage, an observation which correlates with thereported toxicity and carcinogenicity data for chromium(VI) inboth animals and humans.

INTRODUCTION

Chromium(VI) compounds pose a serious occupational healthhazard in terms of both their potential respiratory tract carcinogenicity and their toxic effect on other organs. Extensive epide-

miological evidence has been published on the high incidence ofrespiratory tract cancer in chromate workers in various countries(7, 25). In addition, renal and hepatic damage resulting fromexposure to chromium(VI) compounds has been reported forchromium workers (24). In animals, several studies have reportedthe induction of tumors by chromium(VI) compounds at theinjection and implantation site (24, 25). In dust inhalation studieswith animals, chromium(VI) compounds were reported as lungcarcinogens and as remote-site carcinogens (24). In addition,renal (1-3, 15-17, 19, 23, 26, 30-32) and hepatic (32) damage

has resulted in animals after injection of chromium(VI) compounds by various routes.

The interaction of chromium compounds with nucleic acids isimportant to the mechanism of chromium(VI)-induced carcino

genicity. In vitro, the presence of a rat liver microsomal metabolizing system was necessary in order to observe reaction between chromium(VI) and DNA (or RNA) (35). These studies

1This investigation was supported by Grant BC-320 from the American Cancer

Society and by an A. P. Sloan Research Fellowship.2To whom requests for reprints should be addressed.

Received May 5, 1983; accepted August 22, 1983.

suggested that chromium formed a stable ternary complex withDNA and protein after metabolism of chromium(VI) to chro-mium(lll). Using the alkaline elution technique, DNA-protein crosslinks have been observed upon treatment of various culturedmammalian cells with chromium(VI) compounds (4, 14, 22).These DNA-protein cross-links appeared resistant to repair,

since no decrease in their level was seen 12 to 24 hr afterremoval of chromium(VI) (4, 14). No DNA interstrand cross-links

have been observed in cultured cells treated with chromium(VI)(4,13,14).

The alkaline elution technique (13,14,22) and alkaline sucrosegradients (8, 28, 36) have been used to detect DNA strandbreaks in cultured mammalian cells treated with chromium(VI)compounds. In studies where the toxicity of chromium(VI) wasmeasured, DNA strand breaks were observed only at chro-mium(VI) concentrations which were ~ 100-fold higher than

those which produced a detectable toxic effect on the cells (8,28, 36). Other studies reported that no DNA strand breaks wereobserved by alkaline elution (4) or by alkaline sucrose gradient(5, 8) analysis of cultured cells treated with chromium(VI) compounds. Repair of chromium(VI)-induced DNA strand breaks wasobserved in human embryonic lung fibroblasts (IMR-90 cells) 4

or 12 hr after removal of chromium(VI) (14). Unscheduled DNAsynthesis was observed in cultured human skin fibroblasts (36)and mouse fetal cells (27) exposed to chromium(VI) compounds.An increase in the amount of DNA strand breakage was observedin the presence of a DNA polymerase inhibitor in chromium(VI)-

treated normal and xeroderma pigmentosum human fibroblasts(13). It was concluded that chromium(VI)-induced DNA damage

must be repaired by a system other than the UV excision repairsystem (13).

Since metabolism of chromium(VI) appears to be important tothe interaction of chromium with DNA and since chromium(VI)induced variable DNA damage and repair in different cultured cellsystems, we have studied in vivo DNA damage in various tissuesof rats treated with chromium(VI). We previously reported thatDNA damage occurred in vivo in rat kidney and liver 1 hr afteran i.p. injection of sodium dichromate (34). The present studydescribes the time course for formation and removal of DNAlesions in rat kidney, liver, and lung over a period of 0 to 40 hrafter an i.p. injection of sodium dichromate. The distribution ofchromium in the tissues and in the nuclei isolated from thesetissues was measured 0 to 24 hr after injection using [51Cr]-

chromate. The chromium distribution in these organs is discussed with respect to the observed DNA damage.

MATERIALS AND METHODS

Chemicals. Sodium dichromate (Na2Cr2O7•2H2O) and chromic chloride (CrCI3-6H2O) were purchased from Fisher Chemical Co., Pittsburgh,Pa.; 51Cr, as Na251CrO4in 0.9% NaCI solution, 2.5 /¿gchromium per ml,

1 mCi per ml, was purchased from Amersham, Inc., Arlington Heights,

5662 CANCER RESEARCH VOL. 43

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Chromium(VI)-induced DNA Lesions

III.; lithium dodecyl sulfate was purchased from Bio-Rad Laboratories,Richmond, Calif.; proteinase K was purchased from Boehringer-Mann-

heim, Indianapolis, Ind.; tetraethylammonium hydroxide was purchasedfrom Eastman Chemicals, Rochester, N. Y. Caution: sodium dichromateis a potential carcinogen and should be handled with care.

Nuclei Preparation. Male Sprague-Dawley [CRL:CD(SD)BR]) rats

(Charles River Breeding Laboratories, Wilmington, Mass.) weighing 150to 200 g were given i.p. injections of 0.5 ml of 0.9% NaCI solutioncontaining the sodium dichromate (20 or 40 mg/kg) or chromic chloride(80 mg/kg). Control rats were given i.p. injections of 0.5 ml of 0.9% NaCIsolution. At 0 to 40 hr after injection, the rats were sacrificed bydecapitation, and nuclei from the right renal cortex, the front hepaticlobe, or the whole lung were prepared as described previously (6, 34).In experiments using radioactively labeled chromium, the nuclei werepurified by centrifugation at 130,000 x g through 70% sucrose (w:v) at4° for 90 min using a Sorvall Model AH-627 swinging bucket rotor.

Nuclei were counted after staining with 0.04% trypan blue followed bymicroscopic examination using a hemacytometer.

Alkaline Elution. Alkaline elution was performed according to theprocedure of Kohn ef al. (20) as modified by Ciccarelli and Wetterhahn(6). The amount of DNA in the eluate and on the polyvinyl chloride filterswas determined using the microfluorometric assay, as described previously (6).

Determination of Chromium Levels with Radioactively Labeled

020

8 I2 16Time After Injection (hours)

Chart 1. DNA cross-links in rat kidney nuclei at various times after a single i.p.injectionof sodium dichromate (20 mg/kg). Cross-linkingcoefficientwas calculatedas specified by Ewig and Kohn (12). •,total cross-linking; O, cross-linking aftertreatment of lysates with proteinase K. Bars, S.E. associated with values determined from 3 to 9 rats.

Chromate. Male Sprague-Dawley [CRL:CD(SD)BR] rats (Chartes River

Breeding Labs) weighing 150 to 200 g were given i.p. injections of 0.5ml of 0.9% NaCI solutions containing appropriate doses of cold Na2Cr207mixed with a known amount of Na251CrO<.Typically, 30 to 50 nC\ of 51Cr

were administered per rat in addition to the appropriate dose of coldNa2Cr2O7. One g of lung, liver, and kidney tissue was removed andhomogenized, as specified above for the nuclei preparation, and countedon a Beckman Model 8000 gamma counter (Beckman Instruments, Inc.,Fullerton, Calif.). Sucrose-purified nuclei, prepared as described above,were also counted for 51Cr. After subtracting background cpm and

correcting cpm for decay during the course of the experiment, the knownspecific activity of the injected chromate solution was used to calculateng chromium per g tissue and per 106 nuclei.

RESULTS

Chromate-induced DNA Damage in Rat Tissues. DNA dam

age in rat kidney, liver, and lung following an i.p. injection ofsodium dichromate was measured by the alkaline elution technique on nuclei isolated from these organs. DNA strand breaks,DNA-protein cross-links, and interstrand cross-linking were eval

uated as specified by Kohn ef al. (20).The time course for formation of DNA cross-links in rat kidney

after injection of sodium dichromate (20 mg/kg body weight) ispresented in Chart 1. Within 1 hr after injection, total cross-linking reached a maximum. At 12 hr, total cross-linking de

creased to a lower but significant level which persisted to 40 hr(longer times after injection were not examined). In contrast withthe decreasing trend in cross-linking observed at the 20-mg/kg

dose of sodium dichromate between 4 and 12 hr (Chart 1), thelevel of cross-linking increased between 4 and 12 hr at the 40-

mg/kg dose (Table 1). Upon treatment of the kidney nuclearlysates with proteinase K, DNA-protein cross-links were removedrevealing the presence of interstrand cross-links. Interstrandcross-linking was observed at a constant level 1 through 24 hr

after injection of 20 mg/kg (Chart 1); however, by 40 hr, theinterstrand cross-links were removed. Interstrand cross-linking

was also seen at both 4 and 12 hr after injection of the 40 mg/kg dose (Table 1). No cross-linking was observed in the kidney

1 hr after injection of chromium(lll) chloride at 80 mg/kg, a dose

Table 1Apparent frequencies of DNAlesionsproduced in rat kidney, liver, and lung nuclei after treatment with sodium dichromate or chromic chloride

Strand breaks (radequivalents)Tissue

CompoundKidney

Na2Cr2O7-2H2OCrCI3.6H2OLiver

NaîCr2O7-2H2OLung

Na2Cr2O7-2H2ODose

(mg/kg)20204040802020404020204040Exposure(hr)41241214124121414Withoutprotein

aseK10±4a'"(3)c0*1

(3)5±12'(3)-34+

^(3)0+1V(2)11

+ 6'(6)0±6'(5)32+1"(3)-23+5'(3)7±

4"(4)28± 5e(3)4±

/(3)-18± 3' (3)With

proteinaseK11

± 3"(6)0±1'(3)9+4*(3)-14

± V(3)-6

± ¿(3)15± 3a(3)3± 4'(3)2±

1"(3)11

± 2e(3)-62+15'(3)0+3'

(3)28± 6e (3)Cross-link

coefficientWithout

proteinaseK0.128 ±0.008"(5)0.076

±0.013e(3)0.085±0.008"(3)0.1

53 ±0.006"(3)0.001±0.030'(2)0.289

+ 0.046e(6)0.073±0.008"(5)0.167±0.019e(3)0.1

57 ±0.056"(3)0.01

5 ±0.016'(2)-0.022+ 0.004'(3)0.1

23 ±0.005"(3)0.172 ±0.019" (3)With

proteinaseK0.039±0.006a(3)0.027±0.003"(3)0.024+ 0.019'(3)0.065+ 0.016e(3)0.027

+ 0.006e(3)-0.005+ 0.003'(3)-0.014±0.001'(3)0.017+ 0.014'(3)0.001

+ 0.005'(3)-0.038+ 0.004'(3)0.060±0.005"(3)0.054+ 0.011e (3)

" Mean ±S.E. Calculatedby the equations of Ewig and Kohn (12).bp < 0.1 versus control, t test (38).' Numbers in parentheses,number of rats tested."p < 0.01 versus control, ( test (38).8p < 0.05 versus control, i test (38).'p > 0.1 versus control, t test (38).

DECEMBER 1983 5663

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

M. J. Tsapakos et al.

which is equimolar in chromium to a 45-mg/kg dose of sodium

dichromate (Table 1).The time course for induction of cross-links in rat liver as a

function of time after injection of sodium dichromate (20 mg/kg)is presented in Chart 2. In contrast to kidney, total cross-links in

liver rose sharply to a peak at 4 hr after injection and thendeclined rapidly through 8 hr. The maximum amount of totalcross-linking in the liver was approximately twice that observedin the kidney (Table 1). However, unlike the kidney, cross-linking

had completely disappeared in the liver 36 hr after injection. Theinterstrand cross-linking in the liver reached a maximum value at2 hr after injection but had disappeared by 8 hr. Total cross-

linking in liver nuclear DMA at the higher dose of sodium dichromate (40 mg/kg) remained at approximately the same level at 4and 12 hr after injection (Table 1) in contrast to the decrease incross-linking seen between 4 and 12 hr after injection at thelower dose (20 mg/kg). At the higher dose, no interstrand cross-

linking was observed at 4 hr, and only a low level of interstrandcross-linking was detected 12 hr after injection.

The time course for formation of cross-links in lung after

injection of sodium dichromate is presented in Chart 3. As inboth liver and kidney, significant cross-linking in the lung wasobserved 1 hr after injection, and total cross-linking peaked at 4hr. Little or no cross-linking was observed in lung 1 or 4 hr after

injection of the lower dose (20 mg/kg) (table 1). By 24 hr afterinjection of sodium dichromate (40 mg/kg), total cross-linkinghad decreased to about one-half the maximum value, and thislevel persisted through 36 hr. Interstrand cross-linking in the lung

reached maximum 1 hr after injection and gradually declined to

. 0 30 -

0030-

-005.8 I2 I6 20

Time After Injection (houts)

Chart 2. DNA cross-links in rat liver nuclei at various times after injection ofsodium dichromate (20 mg/kg). See legend to Chart 1 for conditions.

0 after 36 hr. Thus, as with kidney, DNA-protein cross-linkingwas the major cross-linking lesion to persist through 36 hr in the

lung.Only low levels of DNA strand breaks (in the presence or

absence of proteinase K treatment) were observed in rat kidneyand liver at various times after injection of sodium dichromate(20 or 40 mg/kg) (Table 1 and data not shown). No strand breaksin rat lung were observed in the absence of proteinase K digestion through 24 hr; however, a small amount of strand breakage[24 ±3 (S.E.) rad equivalent] was observed 36 hr after injectionof sodium dichromate (40 mg/kg) (Table 1 and data not shown).Upon proteinase K treatment of the lysates, DNA strand breaksin rat lung (maximum of 28 ±6 rad equivalents) appeared at 4to 12 hr after injection of sodium dichromate (40 mg/kg), wereremoved, and then reappeared at 36 hr (33 ±3 rad equivalents)(Table 1 and data not shown). Only low levels of strand breakswere observed in rat lung after injection of sodium dichromate(20 mg/kg) (Table 1). At the 20-mg/kg dose, a maximum of 21

±3 rad equivalents of strand breaks were observed in rat liver1 hr after injection; however, no strand breaks remained by 24or 36 hr (data not shown). In the kidney, DNA strand breaks (12±6 rad equivalents maximum) were observed 4 to 8 hr afterinjection of 20 mg/kg; however, all strand breaks were removedby 12 hr (Table 1), and then strand breaks were observed onlyafter proteinase K treatment at 24 (9 ±1 rad equivalents) and40 hr (14 ±2 rad equivalents) (data not shown). No strandbreaks were observed in rat kidney 1 hr after injection of chro-mium(lll) chloride (80 mg/kg) (Table 1).

Chromium Distribution. The concentration of chromium in ratkidney, liver, and lung tissues was measured after a single 20-or 40-mg/kg injection of Na2Cr2O7 containing Na251CrO4at a

known specific activity. At the 40-mg/kg dose, levels of chromium for liver tissue (3.18 ±0.33% dose administered per gtissue) and kidney tissue (3.41 ±0.29% dose administered perg tissue) peaked 12 hr after injection (Chart 4). In kidney andliver tissue at the 20-mg/kg dose, levels of chromium reachedmaximum at 4 hr after injection (2.42 ±0.40% dose administeredper g kidney tissue; 2.91 ±0.68% dose administered per g livertissue) (Chart 4). A significant amount of chromium was in thetissues by 1 hr (1.73 ±1.16% dose administered per g kidneytissue; 1.77 ±0.74% dose administered per g liver tissue). At a40-mg/kg dose of sodium dichromate for both kidney and liver,

chromium levels in nuclei increased with time after injection

0.20 -

8cOIO-

0.05

8 I2 I6Time After Injection (hours)

Chart 3. DNA cross-links in rat lung nuclei at various times after injection ofsodium dichromate (40 mg/kg). See legend to Chart 1 for conditions.

Chart4. Distribution of chromium in kidney (A)and liver tissue (B)after a singlei.p. injection of sodium dichromate. Rats were given sodium dichromate injectionsof 20 mg/kg (O)or 40 mg/kg (•)spiked with 30 ¿iCiNa251CrO«per rat and sacrificedat the indicated times. Bars, S.E. associated with values determined from 2 to 4rats.

5664 CANCER RESEARCH VOL. 43

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Chromium(VI)-induced DNA Lesions

(Chart 5). At 12 to 24 hr after injection, the chromium levels inliver nuclei were higher than they were in kidney nuclei. At thelower (20-mg/kg) dose of sodium dichromate, the amount of

chromium in both kidney and liver nuclei reached a maximum at4 hr after injection and decreased to a low but persistent levelat 8 through 24 hr (Chart 5).

Chromium distributions in lung tissue and nuclei are presentedin Chart 6. Generally, the levels of chromium in the lung were 5to 10 times lower than those found in kidney and liver. At a 40-

mg/kg dose of sodium dichromate, as with liver and kidney, thelung tissue chromium concentration was maximum 12 hr afterinjection (0.61 ±0.10% dose administered per g tissue) (Chart6A). The lower dose of 20 mg/kg produced maximum chromiumconcentration in lung tissue 1 hr after injection (0.27 ±0.22%dose administered per g tissue). Lung nuclei chromium levels(Chart 6B) generally followed the same trends which were observed in the lung tissue with both doses of sodium dichromate.At the 20-mg/kg dose of sodium dichromate, chromium levels in

lung nuclei peaked 4 hr after injection, decreased through 8 hr,and then increased slightly through 24 hr. At the higher (40-mg/

kg) dose, chromium levels in lung nuclei peaked at 12 hr afterinjection followed by a decline through 24 hr.

The number of nuclei which sedimented through sucrose inthe chromium distribution study remained fairly constant regardless of the source of nuclei and time after injection of sodiumdichromate, indicating that minimal lethal cell damage had oc-

Chart 5. Distribution of chromium in sucrose-purified kidney (A) and liver (B)nuclei after a single i.p. injection of sodium dichromate. See legend to Chart 4 forconditions.

Time Aft«Injtction (hours) Tim«Aflftr Injvcrion (hour!)

Charte. Distribution of chromium in rat lung tissue (A) and sucrose-purifiednuclei (B) after a single i.p. injection of sodium dichromate. See legend to Chart 4for conditions.

curred in any of the organs analyzed under any of the conditionsused in our experiments.

DISCUSSION

We reported previously that chromium(VI) induced DNA-pro-tein cross-links and low levels of DMA strand breakage in rat

liver and kidney 1 h after i.p. injection of sodium dichromate (34).The time course for formation and removal of DMA strand breaks,DNA-protein cross-links, and interstrand cross-links has been

presented for kidney, liver, and lung after exposure of rats tosodium dichromate. Chromium(VI) induced significant levels ofDNA-protein and interstrand cross-linking in all 3 organs withinthe first 4 hr after injection. The major form of cross-links wasDNA-protein cross-links. Other studies (4, 14, 22) of chro-mium(VI)-induced DNA damage in cultured mammalian cells reported the occurrence of DNA-protein cross-links but not interstrand cross-links. Formation of DNA-protein cross-links in rat

kidney and lung appeared to saturate and then gradually declinedto a lower but persistent level. The level of interstrand crosslinks in rat kidney and lung remained fairly constant between 1and 12 to 24 hr after injection, but in contrast to DNA-proteincross-links, the interstrand cross-links were not persistent.

These results may indicate that a steady state situation existsbetween formation and removal of interstrand and DNA-proteincross-links which directly involve chromium. It is possible thatthe persistent DNA-protein cross-links result from cross-linking

between DNA and repair enzymes which were activated to repairDNA lesions induced by chromium(VI). The persistence of someof the DNA-protein cross-links may indicate that in kidney and

lung this DNA lesion is not recognized by cellular repair proteinsor that the various cell types in kidney and lung have differentcapacities to repair the chromium(VI)-induced DNA-protein cross

links. A recent study showed that in vivo repair, as measured byunscheduled DNA synthesis in mouse skin treated with variouschemical carcinogens, was 3- to 5-fold more active in the mouse

epithelial cells than in the dermal fibroblasts (18).The trends in the cross-linking time courses for kidney and

lung can be contrasted with that for liver after exposure tochromium(VI). In liver, DNA-protein cross-links are formed within4 hr; however, 90% were removed by 8 hr, and no cross-links

persisted in this organ. It also appears that interstrand crosslinks are repaired faster in liver than in kidney and lung, since inliver interstrand cross-links were completely removed 8 hr follow

ing injection. These results may indicate that rat liver cells, whichpossess high metabolic activity, may be more efficient at repairing chromium(VI)-induced DNA damage than are kidney or lung

cells.The results of the chromium distribution studies indicate that

significant levels of chromium accumulate in rat kidney, liver, andlung at relatively short times following an i.p. injection of sodiumdichromate spiked with radioactive chromium(VI). These findingsconfirm results of other studies of chromium distribution in ratsafter i.v. administration of sodium chromate (21, 29) and s.c.administration of potassium dichromate (37). The higher chromium levels were found in the liver and kidney, with the liverhaving the highest levels at all times for both doses of chro-

mium(VI) examined. Levels of chromium in lung tissue wereapproximately 10 to 20% of those observed in liver at all timesfor both doses. The very low level of chromium distributed to thelung tissue and nuclei after the 20-mg/kg dose probably accounts

DECEMBER 1983 5665

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

M. J. Tsapakos et al.

for the lackof observableDNAdamageundertheseconditions.The trends in chromium levels in rat liver and kidney nuclei

varied depending on the dose that was administered. DNAdamage in the form of DNA cross-linking generally correlated

with the chromium levels in liver and kidney nuclei. Maximumdamage as DNA-protein cross-linking in liver nuclei occurred at

4 hr after injection of sodium dichromate (20 mg/kg), correlatingwell with the maximum nuclear chromium level at this time anddose. In kidney nuclei at this dose, the correlation was less clear,since chromium levels dropped after 4 hr; yet DNA-protein cross-linking persisted at a high level through 8 hr. By 24 hr, cross-

linking had been significantly reduced in both liver and kidney,correlating with a decrease in nuclear chromium levels at thistime and dose. In both kidney and liver, DNA cross-links and

nuclear chromium levels were higher at the lower dose (20 mg/kg) than at the higher dose (40 mg/kg) 4 hr after injection.However, by 12 hr after injection DNA cross-links and nuclearchromium levels were higher at the 40-mg/kg dose than at the

lower dose. In contrast to the decrease in nuclear liver andkidney chromium levels after 4 hr at the 20-mg/kg dose of sodium

dichromate, there is a steady increase in chromium levels withtime after exposure to 40 mg/kg in these organs. In kidney, thisincrease correlates with increased DNA-protein and interstrandcross-linking observed between 4 and 12 hr after injection. Inliver nuclei, which were found to contain much higher levels ofchromium than kidney nuclei 12 hr after injection, DNA-proteincross-linking reached a plateau at 4 through 12 hr. It is possible

that repair mechanisms which are functional in the liver at thelower dose may be either inhibited or saturated at the higherdose. Chromium may be interacting with nuclear proteins, suchas repair enzymes, either inhibiting or blocking their function.

It appears that lung and kidney are more sensitive than liverto the low levels of chromium in the nucleus after injection ofsodium dichromate. Lung nuclear chromium levels at saturationof DNA-protein cross-linking (approximately 10 to 17 ng chromium per 106 nuclei) were close to kidney nuclear chromiumlevels at saturation of cross-linking (approximately 15 to 25 ngchromium per 106 nuclei). However, the corresponding value for

liver was much higher (approximately 60 to 70 ng chromium per106 nuclei). In contrast to chromium(VI), chromium(lll) producedno detectable strand breaks or cross-links in the rat kidney 1 hr

after injection. Other studies have shown that after s.c. injectionof chromium(lll) in rats, 10- to 100-fold lower levels of chromium

are detected in kidney, liver, and lung tissues than after injectionof chromium(VI) at a dose equimolar in chromium (37). In addition,lower levels of chromium were detected in liver nuclei up to 12hr after i.v. injection of chromium(lll) compared to an equimolarinjection of chromium(VI) (29). These results correlate with thegreater kidney and liver toxicity of chromium(VI) compared tochromium(lll) after i.p. injection in rabbits (23,32). No DNA crosslinks were observed in human lung fibroblasts or mouse L1210leukemia cells treated with chromium(lll) in contrast to chro-mium(VI) which produced DNA-protein cross-links (14). However,DNA cross-links were observed upon treatment of isolated nuclei

with chromium(lll) (14). This result supports the correlation ofDNA damage with nuclear chromium levels which we found invivo.

For kidney, liver, and lung, there may be a relationship betweenthe persistence of cross-linking and organ toxicity and carcino-

genicity. Chromium(VI) has been shown to induce kidney lesionsin monkeys (17), dogs (15, 31), rabbits (23, 26, 32), and rats (1-

3,16,19,30) as well as in enrómate workers (24). Chromium(VI)induced respiratory tract cancer in chromate production workers(7, 25) and was implicated as a lung carcinogen in rats andhamsters (24). In contrast, liver damage in humans and animalshas not been extensively documented (24, 32).

A relationship between DNA cross-linking and cytotoxicity has

been reported for various drugs. The sensitivity of 2 human coloncarcinoma cell lines to 1-(2-chloroethyl)-3-(4-f/-ans-methylcyclo-

hexyl)-1 -nitrosourea was correlated to the extent of cross-linkingafter drug treatment (33). The cytotoxicity of haloethylnitrosou-reas in V-79 Chinese hamster cells was related to their ability tocross-link DNA (10). Chloroethylnitrosoureas induced morecross-links in a sensitive SV40 transformed human embryo cell

line than in a less sensitive normal human fibroblast cell line (9).The cytotoxicity of cyclophosphamide derivatives in mouseL1210 cells correlated with DNA cross-link formation (11). DNAcross-links which were produced by c/s- and frans-dichloro-diammineplatinum(ll) in mouse L1210 cells and V-79 Chinese

hamster cells correlated with the observed cytotoxicity of theplatinum complexes in these cells (39, 40). Another metal carcinogen, nickel carbonate, induced DNA cross-links in rat kidney

nuclei in vivo (6).In conclusion, chromium(VI) rapidly induced significant levels

of cross-linking in rat kidney, liver, and lung. DNA-protein cross

links persisted 36 to 40 hr after injection in rat kidney and lung,yet had been repaired in liver by 36 hr. These data, in additionto the organ chromium distribution data, suggest that lung andkidney are more sensitive than liver to chromium-induced DNAdamage. This correlates with the chromium(VI) toxicity datareported for both animals and humans.

REFERENCES

1. Saines, A. D. Cell renewal following dichromate induced renal tubular necrosis.An enzyme histochemical study. Am. J. Pathol., 47: 851-876, 1965.

2. Berndt, W. 0. The effect of potassium dichromate on renal tubular transportprocesses. Toxicol. Appi. Pharmacol., 32: 40-52, 1975.

3. Biber, T. U., Mylle, M., Baines, A. D., Gottschalk, C. W., Oliver, J. R., andMacDowel, M. C. A study by micropuncture and microdissection of acute renaldamage in rats. Am. J. Med., 44: 664-705, 1968.

4. Brambilla, G., Sciaba, L.. Carlo, P., Finollo, R., Farina, A., and Parodi, S. DNAcross-linking in mammalian cells treated with potassium dichromate. Proc. Am.Assoc. Cancer Res., 21: 98, 1980.

5. Casto, B. C., Pieczynski, W. J.. Nelson, R. L., and DiPaolo, J. A. In vitrotransformation and enhancement of viral transformation with metals. Proc.Am. Assoc. Cancer Res., 17: 12, 1976

6. Ciccarelli, R. B., and Wetterhahn, K. E. Nickel distribution and DNA lesionsinduced in rat tissues by the carcinogen nickel carbonate. Cancer Res., 42:3544-3549,1982.

7. Doll, R. Problems of epidemiological evidence. Environ. Health Perspect., 40:11-20,1981.

8. Douglas, G. R., Bell, R. D. L, Grant, C. E., Wytsma, J. M., and Bora, K. C.Effect of lead chromate on chromosome aberration, sister-chromatid exchangeand DNA damage in mammalian cells in vitro. Mutât.Res., 77:157-163,1980.

9. Erickson, L. C., Bradley, M. O., Ducore, J. M., Ewig, R. A. G., and Kohn, K.W. DNA cross-linking and cytotoxicity in normal and transformed human cellstreated with antitumor nitrosoureas. Proc. Nati. Acad. Sei. U. S. A., 77: 467-

471,1980.10. Erickson, L. C., Bradley, M. O., and Kohn, K. W. Measurements of DNA

damage in Chinese hamster cells treated with equitoxic and equimutagenicdoses of nitrosoureas. Cancer Res., 38: 3379-3384, 1978.

11. Erickson, L. C., Ramonas, L. M., Zaharko, D. S., and Kohn, K. W. Cytotoxicityand DNA cross-linking activity of 4-sulfidocyclophosphamides in mouse leukemia cells in vitro. Cancer Res., 40: 4216-4220, 1980.

12. Ewig, R. A. G., and Kohn, K. W. DNA-protein cross-linking and DNA interstrandcross-linking by haloethylnitrosoureas in L1210 cells. Cancer Res., 38: 3197-3203, 1978.

13. Fornace, A. J. Detection of DNA single-strand breaks produced during therepair of damage by DNA-protein cross-linking agents. Cancer Res., 42: 145-149, 1982.

14. Fornace, A. J., Seres, D. S., Lechner, J. F., and Harris, C. C. DNA-proteincross-linking by chromium salts. Chem.-Biol. Interact., 36: 345-354,1981.

5666 CANCER RESEARCH VOL. 43

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

15. Hepler, O. E., and Simonds, J. P. Experimental nephropathies IV. Glycosuriain dogs poisoned with uranyl nitrate, mercury bichloride and potassium dichro-mate. Arch. Pathol., 41:42-49, 1946.

16. Hirsch, G. H. Differential effect of nephrotoxic agents on renal organic ¡ontransport and metabolism. J. Pharmacol. Exp. Then, 786: 593-599,1973.

17. Hunter, W. C., and Roberts, J. M. Experimental study of the effects ofpotassium dichromate on the monkey's kidney. Am. J. Pathol., 9: 133-147,

1933.18. Ishikawa, T., Kodama. K., Ide, F., and Takayama, S. Demonstration of in vivo

DNA repair synthesis in mouse skin exposed to various chemical carcinogens.Cancer Res., 42: 5216-5221,1982.

19. Kirschbaum, B. B., Sprinkel, F. M., and Oken, D. E. Proximal tubule brushborder alterations during the course of chromate nephropathy. Toxicol. Appi.Pharmacol., 58.' 19-30, 1981.

20. Kohn, K. W., Ewig, R. A. G., Erickson, L. C., and Zwelling, L. A. Measurementof strand breaks and cross-links in DNA by alkaline elution. In: E. C. Friedbergand P. C. Hanawalt (eds.), DNA Repair: A Laboratory Manual of ResearchProcedures, Vol. 1, Part B, pp. 379-402. New York: Marcel Dekker, Inc.,

1981.21. Langärd,S. The time-related subcellular distribution of chromium in the rat

liver cell after intravenous administration of Na251CrO4.Biol. Trace ElementRes., 1: 45-54, 1979.

22. Lechner, J. F., Haugen, A., Tokiwa, T., Trump, B. F., and Harris, C. C. Effectsof asbestos and carcinogenic metals on cultured human bronchial epithelium(abstract). In: Meeting on Carcinogenesis Studies Using Cultured HumanTissues and Cells, September 20 to 24,1982, Aspen, Colo.

23. Mathur, A. K., Chandra, S. V., and Tandon, S. K. Comparative toxicity oftrivalent and hexavalent chromium to rabbits. II. Morphological changes insome organs. Toxicology, 8: 53-61, 1971.

24. National Institute of Occupational Safety and Health. Biological effects ofexposure. In: Criteria for a Recommended Standard: Occupational Exposureto Chromium(VI), pp. 23-121. Washington, D. C.: U. S. Department of Health,Education, and Welfare, 1975.

25. Norseth, T. The carcinogenicity of chromium. Environ. Health Perspect., 40'121-130,1981.

26. Ophüls,W. Experimental nephritis in rabbits by subcutaneous injections ofchromâtes. Proc. Soc. Exp. Biol. Med., 9: 13, 1911.

27. Raffetto, G., Parodi, S., Parodi, C., DeFerrari, M., Troiano, R., and Brambilla,G. Direct interaction with cellular targets as the mechanism for chromiumCarcinogenesis. Tumori, 63: 503-512,1977.

Chromium(VI)-induced DNA Lesions

28. Robison, S. H., Cantoni, O., and Costa, M. Strand breakage and decreasedmolecular weight of DNA induced by specific metal compounds. Carcinogenesis (Lond.), 3: 657-662,1982.

29. Sayato, Y., Nakamuro, K., Matsui, S., and Ando, M. Metabolic fate of chromiumcompounds. I. Comparative behavior of chromium in rat administered withNa251CrO4and 51CrCI3.J. Pharm. Dyn., 3:17-23,1980.

30. Schwartz, R. H., Lewis, R. A., and Schenk, E. A. Tamm-Horsfall mucoprotein.III. Potassium dichromate-induced renal tubular damage. Lab. Invest., 27:214-217,1972.

31. Simonds, J. P., and Hepler, O. E. Experimental nephropathies I. A method ofproducing controlled selective injury of renal units by means of chemical agents.Arch. Pathol., 39: 103-108, 1945.

32. Tandon, S. K., Saxena, D. K., Gaur, J. S., and Chandra, S. V. Comparativetoxicity of trivalent and hexavalent chromium: Alterations in blood and liver.Environ. Res., 75: 90-99, 1978.

33. Thomas, C. B., Osieka, R., and Kohn, K. W. DNA cross-linking by in vivotreatment with 1-<2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosoureaof sensi

tive and resistance human colon carcinoma xenografts in nude mice. CancerRes., 38:2448-2454,1978.

34. Tsapakos, M. J., Hampton, T. H., and Jennette, K. W. The carcinogen chromateinduces DNA cross-links in rat liver and kidney. J. Biol. Chem. 256- 3623-

3626,1981.35. Tsapakos, M. J., and Wetterhahn, K. E. The interaction of chromium with

nucleic acids. Chem.-Biol. Interact., in press, 1983.36. Whiting, R. F., Stich, H. F., and Koropatnick, D. J. DNA damage and DNA

repair in cultured human cells exposed to chromate. Chem.-Biol. Interact., 26:267-280, 1979.

37. Yamaguchi, S., Sano, K., Shimojo, N., Hirota, Y., and Kano, K. On theelimination of chromium compounds in rats. In: Proceedings Chromâtes Sym-posium-80, September 16 to 18, 1980, pp. 32-42. Rockville, Md., 1981.

38. Young, H. D. Statistical Treatment of Experimental Data, p. 160. New York:McGraw-Hill Book Co., Inc., 1962.

39. Zwelling, L. A., Anderson, T., and Kohn, K. W. DNA-protein and DNA inter-strand cross-linking by c/s- and rrans-platinum(ll) diamminedichloride in L1210mouse leukemia cells and relation to cytotoxicity. Cancer Res., 39: 365-3691979.

40. Zwelling, L. A., Bradley, M. 0., Sharkey, N. A., Anderson, T., and Kohn, K. W.Mutagenicity, cytotoxicity and DNA cross-linking in V79 Chinese hamster cellstreated with c/s- and frans-Pt(ll) diamminedichloride. Mutât. Res., 67: 271-280, 1979.

DECEMBER 1983 5667

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1983;43:5662-5667. Cancer Res   Michael J. Tsapakos, Thomas H. Hampton and Karen E. Wetterhahn  in Rat Kidney, Liver, and LungChromium(VI)-induced DNA Lesions and Chromium Distribution

  Updated version

  http://cancerres.aacrjournals.org/content/43/12_Part_1/5662

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/43/12_Part_1/5662To request permission to re-use all or part of this article, use this link

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from