Perspectives on the mechanism of action of the splenic toxicity of aniline and structurally-related compounds

Fd Chem Toxtc Vol 25, No 8, pp 619-626, 1987 Pnnted m Great Bntam All rights reserved

0278-6915/87 $3 00+0 00 Copynght© 1987 Pergamon Journals Ltd

Review Section PERSPECTIVES ON THE M E C H A N I S M OF ACTION OF THE SPLENIC TOXICITY OF ANILINE AND STRUCTURALLY-RELATED COMPOUNDS J S BUS* and J A PoPP Chemical Industry Institute of Toxicology, P O Box 12137, Research Triangle Park, NC 27709, USA (Recewed 5 January 1987) Summary--Anlhne and several structurally-related aromatic amines produce spleen tumours m rats gwen high doses of compound m 2-year bloassay studies Evaluauon of the pathogenesls of the splemc lesions and characterization of the dlsposiUonof radlolabelled amhne mammals suggests that the spleen tumours may be a secondary response resulting from chemically-mediatederythrocyte toxicity It is proposed that compound-derived toxicaty to erythrocytes results m scavengingof damaged red blood cells by the spleen, lnlUatang a senes of events which may contribute to the development of spleen tumours These events potentially include (1) specaficaccumulauon of the parent compound or toxic metabohtes(s) carried to the spleen by erythrocytes, 00 deposition of erythrocytlc debris, parUcularly iron, which may catalyse Ussue-damagmgfree-radical reactions, and (nl) reduction of splemc hyperplasla resulting from erythrocyte overload Linkage of the splenic tumongemclty of these aromatic amines to an mmal toxic event m the erythrocyte suggests that the carcmogemclty of such compounds may be determined by a definable threshold dose, i e the events leading to the carclnogemcay are not mmated until the capacity of the red blood cell to cope with the toxic result is exceeded

Introduction Of the many chemicals evaluated m chrome ammal bloassay programs, very few have produced toxicity primarily m the spleen. All chemicals which cause this selective and relatwely rare organ toxicity are structurally related to amhne, which has been shown to produce splenic toxicity m Fischer 344 rats m two animal bmassays (Chemical Industry InsUtute of Toxicology (CIIT), 1982; National Cancer Institute (NCI), 1978). Other agents identified as producing spleen-directed toxicity are the chemical ratermediates, p-chloroamhne (NCI, 1979a), azobenzene (NCI, 1979b) and o-toluldme (NCI, 1979c), the sulphone drug dapsone (NCI, 1977) and the pigment D & C Red No 9 (NCI, 1982) The common structural element in this senes of compounds is that they either contain free aromatic amine functional groups or have azo hnkages which are readily metabolized to free amines in biological systems (Fig 1) Selective splenic toxicity ~s an unusual toxic response m animal chronic toxicity studies. There is a clear need to understand the potential importance of this response with respect to human exposure to these compounds. The purpose of this rewew, therefore, ms to put th~s phenomenon into perspecUve by bringing together an evaluaUon of the pathogenes~s of the splemc lesmns with other data obtained from genotoxicity, metabohsm and pharmacokinet~c studies.

*Current address" The Upjohn Company, Pathology/ Toxicology Department, 301 Henrietta Street, Kalamazoo, MI 49007, USA Abbrematwns" CIIT = ChemmcalIndustry Institute of Toxicology; NCI =Natmnal Cancer InsUtute; SCE sister

chromatid exchanges.

Although the primary emphasis of this review IS directed to anlhne, which has been the most extenswely stud~ed compound m this series of splenotox~c chemicals, the close simllanty between the splemc toxicity of amhne and the tox~ctty of the other compounds suggests a common toxic mechamsm

Pathogenesis of the splenic toxicity An exammaUon of the tumour modence data derived from the spleens of Fischer 344 rats chromcally treated with aniline and its structurally related compounds reveals both a dose-dependent and sex-

-NO,O.O,H. Dapsone (4,4'-suLphonyLd,onILlne) NH2

o-Tolu,dlne

NHz

©

Aniline NH2

Cl p - Chtoroan,L,ne

:°

o,.,o DS=C Red No 9

Fig ] Structures of compounds that have been =dentlficd as splemc carcinogens m chromc aroma] b~oassays

619

620

J S BUS a n d J A PovP

Table 1 Incidenceof spleen tumours m male and femalerats treated with andme and structurally-related compounds m the &et for about 104 wk Tumour modence (%) Compound and dose* Males Females Amlme~" 0 mg/kg 10 mg/kg (0 02%) 30 mg/kg (0 06%) 100 mg/kg (0 20%) 0 3%~ 06%~:

0 0 13 37 8 66 0 760

0 0 0 13 20 120

p -Chloroamllnet 0 025% 0 050%

0 20 4

20 20

o-Tolmdme? 0 30% 0 60%

38 8 16 7

26 5 38 8

Dapsonet 0 06% 0 12%

18 0 50 0

0 0

Azobenzenet 002% 0 04%

102 30 6

80 34 0

D & C Red No 9t:~ 0 10% 0 30%

20 52 0

0 0

*Dose of amhne HCI expressed as mg/kg/day in the diet with approximate percent m the diet shown m parentheses Doses of other agents are expressed as percentage compound m the diet l'The tumour data were obtained from the followingsources amhne (CIIT, 1982, NCI, 1978), p-chloroandme (NCI, 1979a), otolmdme (NCI, 1979c), dapsone (NCI, 1977), azobenzene (NCI, 1979b) and D & C Red No 9 (NCI, 1982) ~:A re-evaluatmn of the tumour pathology data from the National Cancer Institute sponsored studms ymlded a tumour incidence of 27% and 60% for the low- and h]gh-dose andme groups, respectively (4% and 10% for corresponding female dose groups) and 0% and 51% for the respeetwe low- and lugh-dose D & C Red No 9 males and 0% for both female dose groups (Wemberger et al 1985) Spleen tumour incidences among controls were zero m all cases related response with respect to splenic cancer (Table 1) Aniline, p-chloroanihne, dapsone, azobenzene and D & C Red N o 9 all produced dose-dependent increases in splenic neoplasms ( G o o d m a n et al 1984). o-Tolmdlne, which did not exhibit a clear dose response, also reduced significant tumour formaUon in other organs, particularly those of the abdominal cavity and therefore the site of ongln of the splenic tumours associated with o-toluldme treatment is obscure ( G o o d m a n et al 1984) The tumorigenic response induced by several of these compounds appears to be non-linearly related to dose In the case of aniline, where two chronic ammal bioassays in the same strain of rats provide data over a broad dose range, no tumours were observed when anihne was fed in the diet at a dally dose of 10 mg/kg (approximately 0 02% in the diet, CIIT, 1982) A threefold increase in dose (30 mg/kg, approximately 0 06% in the &et) produced only a few tumours of the spleen. An additional threefold increase in dose (100 mg/kg, approximately 0 2% in the diet), however, resulted in a &sproportaonate 29-fold increase in tumour incidence Increasing the dietary concentration to 0 3% further increased the tumour incidence At dietary concentrations above 0.3% the increase in tumour incidence is less dramatic (Fig 2) Similar types of non-linear responses

8O 70

E 60

o

~

3o

~ 20

I-I

lO o

01

o2 03 o4 Anitlne in diet (%)

05

06

F~g 2 Splemc tumour incidence m rats gwen dietary amhne The lower three concentratxons were fed m the CIIT study (CIIT, 1982) whale the two higher concentrations were fed m the NIH study (NCI, 1978) also were observed in bloassays of p-chloroamhne andD&C Red N o 9 With the exceptions of o-toluidine and azobenzene, a clear sex-dependent response also was observed in the splenic tumongenicity of these compounds N o spleen tumours were seen in female rats treated with either of two dietary treatments of D & C Red N o 9 dye (NCI, 1982) or dapsone (NCI, 1977) Female rats treated with p-chloroamhne (NCI, 1979) or anlhne (CIIT, 1982, N C I , 1978) exhibited markedly diminished or absent tumorigenic responses relative to comparably treated male rats It is interesting to note that a sex-related t u m o n g e m c response became apparent with both o-toluldlne and azobenzene when t u m o u r data from both the spleen and peritoneal cavity were pooled ( G o o d m a n et al 1984). The ability of these compounds to induce spleen tumours is also species specific Both sexes of B6C3F~ mice have also been evaluated concurrently with all of the compounds described above, and in every instance the B6C3FI mouse &d not develop spleen tumours Splenic fibrosarcomas were the prevalent tumour type observed m the animal bioassays, although haemang~osarcomas were also noted ( G o o d m a n et al 1984; Weinberger et al 1985). In addition to tumours, capsular and parenchymal fibrosis has been reported m spleens of animals killed at the end of the 2-year bioassay period ( G o o d m a n et al. 1984, Weinburger et al 1985) The fibrotlc reactions ranged from focal to diffuse and were frequently assocmted with areas of stromal hyperplasia The intimate association of the areas of fibrosis with the splenic sarcomas has led to the suggestion that these lesions may be preneoplastm ( G o o d m a n et al 1984, Wemberger et al 1985) Fool of mature fat cells within the fibroUc areas of the spleen have also been noted in several studies ( G o o d m a n et al 1984; Weinberger et al 1985) This observalaon has resulted In the hypothesis that the fatty lesion may originate from a chemically-induced transformation of splenic mesenchymal cells (Wemberger et al 1985)

Splemc tox~oty of amhne compounds Because of the potential role of red-blood-cell destruction m the splemc toxicity of amllne and structurally related compounds, the accumulation of haemosldenn, a degradatwe product of red cells, has recewed special attention An evaluation of the amounts of haemos~denn accumulated m spleens of rats from the NCI bloassay of amhne (1978) and D & C Red No 9 (1982) suggested that the presence of this pigment &d not correlate w~th tumour incidence, a high incidence of haemosldenn deposits (75% to 100%) was found in control and both the low and high chemical dose groups (Wemberger et al 1985) However, ewdence of a dose response has been found in splemc haemos~denn accumulation m rats examreed from the anlhne bloassay sponsored by CIIT (1982), m which all the doses used were lower than those used m the NCI study of andme (Table 1) The mcadence of haemosldenn classified as moderate to severe ranged from 1% m control male rats to 14%, 52% and 69% m the 10, 30 and 100 mg/kg body weight/day dose groups respectively The potentml relationship of the various nonneoplastic lesions to the splenic tumours has received extensive speculauon (Goodman et al 1984; Wemberger et al 1985) The hypothesized pathogenesls of the lesions as published in the literature is based on the lesions present after 2 years of chemical exposure The need for a chronic study with serial kflhngs to study the progressive development of the splemc les~ons has been stressed by Wemberger et al (1985) The bloassay ofandIne sponsored by the CIIT (1982) included kdls at 6, 12 and 18 months m ad&Uon to the final at 24 months. Capsullt~S occurred as early as 6 months and was characterized by a mononuclear cell infiltration into the splemc capsule Th~s inflammatory response was associated with thickening of the capsule and capsular fibrosis Capsular cysts were a late developing les~on not being found before 18 months Prohferatwe lesions outside the splemc capsule were not noted before 18 months At this time, small focal lesions were characterized by proliferation of spindle-shaped stromal cells with varymg amounts of collagen Similar but larger les~ons were noted by 24 months when turnouts were first noted The ongm of the stromal hyperplasm could not be determined in all sections although m some cases the early prohferatlve lesion appeared to begin m the subcapsular red pulp Tlus location suggests that the prohferatwe les~ons in the splemc parenchyma are arising from the earher prohferatwe les~ons m the splemc capsule The spectrum of lesions in the splenic parenchyma at 24 months and the slmdanty to the smaller les~ons at 18 months strongly suggests that the tumours are arising from the preexisting non-neoplastic prohferat~ve les~ons m the splenic red pulp The bloassay of anihne (CIIT, 1982) also provided observations consistent w~th the well known acute toxicity of this compound m the erythrocyte (for rewew, see K~ese, 1974) Haematology stu&es conducted as part of the b~oassay in&cated that the haematocnt, haemoglobin and erythrocyte count all were reduced in 30 and 100 mg/kg body weight/day dose groups after 52, 78, and 104 wk of the study, while the reaculocyte count was elevated in the high-dose rats after 26, 52, 78 and 104 wk. Andlne-

621

&retted erythrocyte toxicity was generally less severe m female rats with respect to all of the above erythrocyte parameters The spectrum of toxic effects noted with amhne and other splenotoxlc compounds has resulted m the suggestion of several potential mechanisms by wbach these agents may selectively damage the spleen From an evaluation of the pathology data denved from the bloassays of andlne, p-chloroanlhne, azobenzene, o-toluldme, dapsone and D & C Red No 9, Goodman et al (1984) suggested that spleen toxaclty may ongmate from an lmtlal toxicity to the erythrocytes. Because a major function of the spleen is to remove aged or damaged erythrocytes, chemically-induced toxicity m these cells would result in their accumulation vathm the spleen Goodman et al suggested that this sequestration of erythrocytes by the spleen could result m haemosldenn depomlon m the spleen, and coupled with potentially enhanced delivery of toxic metabohtes earned to the spleen m the scavenged red blood cells, lead to the development of splenic fibrosis and the subsequent formaUon of fibrosarcomas This hypothesis was m close agreement with one put forward by Bus (1983) based upon studies of the dlspostlon of 14C-amhne hydrochlonde (see below), and is substantiated by the haematology data and the dose-dependent splemc haemosldenn accumulation noted m the chromc bloassay (CIIT, 1982) Recently, Welnberger et al (1985) suggested an alternative mechamsm &ffenng shghtly from that proposed by Goodman et al (1984) and Bus (1983) After a re-evaluation of the bioassay data from amhne and D & C Red No 9 (NCI, 1978 & 1982), these mvesUgators proposed that acute vascular congestion resulting from splenic scavenging of chemically-damaged erythrocytes may be an important lmtml toxic lesion to the spleen The vascular congestion would lead to splemc haemorrhage, format~on of fibrous Ussue mass, and again m conjunction with accumulation of toxic chemical metabohtes within the spleen (derived from scavenged erythroeytes), transformation of mesenchymal cells of the spleen These transformed cells were proposed to result m the expression of splemc fibrosarcomas and a vanety of other lesions, such as fatty metamorphosis, noted in tumour-beanng ammals Haemosidenn was not thought to be critical to the development of splemc tumours since these investigators obtained no evidence that either amhne or D & C Red No 9 increased mtrasplemc accumulation of ~ron-contalning p~gment Ttus observation stands m contrast, however, to observations made from the andme bloassay conducted by the CIIT, (1982) m which a dose-dependent accumulation of haemosldenn was noted The understanding of the potential mechanism of the spleen-dwected toxloty of amhne has been significantly advanced by the detaded evaluations of the pathogenesls of the lesions described above However, several additional types of studies have contributed to furthenng the hypothesis that the splemc toxicity of these compounds may be medmted through an mmal interaction w~th erythrocytes First, stu&es have been conducted to determine the potentml for the splenotoxlc agents to produce geno-

622

J S Bus and J A PoPP

toxicity m a vanety of test systems. Secondly, extenswe &sposmon studies have been conducted with amhne, which has served as a useful model compound to provade addmonal insight mto the potential mechanism of the spleen-specific toxlclty of these

agents. Genotoxieity of aniline and related compounds An ~mportant question w~th respect to the splemc carcmogenicity of these chemicals is whether the tumongemc response rmght be medmted by a &rect mteractmn of the chemical or assocmted metabohte(s) wRh D N A m the spleen, or whether the response might be hnked to other potentml cytotox~c responses m the spleen caused by chemical exposure9 To date, stu&es evaluating the genotoxlclty of the splemc carcinogens in bacterial mutagemclty systems have prowded negatwe or inconclusive results Anihne was not mutagemc m Salmonella tester strains, even m the presence of metabohc activaUon (for summary of study results, see Wdmer et al 1981) No mutageniclty was detected m slmdar systems when p-chloroamhne, o-toimdme (Rosenkranz & Pomer, 1979), dapsone (Peters et al 1978), and D & C Red No 9 (Prasad, 1970) were tested In ad&tion to the bacterial test system results, amhne did not increase s~ster chromatld exchanges (SCE) in an m w t r o human fibroblast culture (Wdmer et al 1981) Increased SCE were reported with anihne in a Chinese hamster fibroblast hne, although no dose response was apparent and the length of the incubatmn penod (26 hr) may have contnbuted to the accumulation of spontaneously ox~&zed metabohtes (Abe & Sasakl, 1977). Wdmer et al (1981) evaluated the abd~ty of several putatwe metabohtes of anlhne to produce SCE in human fibroblast cultures Only o-amlnophenoi produced a dose-dependent increase m SCE, although the mcrease reported was approximately 366-fold less than that observed wath the posmve control agent, triethylenemelamme Inductmn of SCE by amllne and several other aromaUc

amines and azo denvatives after m wvo treatment of mice has been examined by Paro& et al (1983). These Investigators concluded that SCE producuon did not correlate well with oncogenic potential for aromatic amines and azo derivatives In other m wtro studies, aniline &d not cause chromosomal damage in V-79 Chinese hamster cells incubated in the presence of a rat liver mlcrosomal activating system (Swenberg et al 1976) As measured by an alkaline elutlon technique, anlhne induced D N A fragmentation m liver and kidney of rats admlmstered single doses of anlhne (Parodl et al 1982) D N A damage was not observed in the spleen, although mulUply-dosed ammals were not evaluated If lesmns induced m the spleen by aniline and =ts congenors are m part due to the accumulation of damaged erythrocytes, as suggested above, multiple doses of compound may be reqmred to produce sufficient red blood cell injury to ehclt detectable splemc macromolecular injury In summary, the lack of genotoxlclty of amhne and structurally related chemicals suggests that the tumorlgemclty of these agents is not assocmted w~th a direct interaction of the compounds or their metabohtes with the D N A of the spleen Rather, the results suggest that "ep~genetlc" or cytotox~c events may play a crmcal role m tumongemc actlwty Further studies will be required, however, to evaluate, more completely, the genotoxlc potentml of these agents in the spleen, and m particular, to consider possible genotox~c responses m animals recewmg multiple exposures to the chemicals

Relationship of the disposition of aniline to spleendirected toxicity Aniline 1s extenswely metabolized, upon systemic absorption, to a vanety of oxidized products Hydroxylatlon of the aromatic nng, forming isomeric phenol metabohtes, represents a major route of amline metabohsm (Fig 3, Kao et al. 1978, Parke, 1960,

Liver

Erythrocyte

N=0 Met-Hb Oxy-Hb HN-OH

c5--

-%

I

Recluctose

1---

NH2

Is)

NH~ •

--"

OH /

I

H

0 . )1.

R

0 II

R=sulphate, gLucuronide Fig 3 Relationships of aniline metabohsm m the hver and erythrocyte The common metabohte phenylhydroxylamme is produced in small amounts m the liver and is taken up by the erythrocytes where extenswe oxldatmn to mtrosobenzene occurs

Splenic toxicity of anlhne compounds Smith & Williams, 1949) The hydroxylated metabohtes are rapidly metabohzed to sulpbates or glucuromdes, and excreted into unne as water-soluble metabohtes Aniline also undergoes extensive Naeetylation, coupled with rang hydroxylatlon, In several ammal species (Fig 3, Kao et al 1978) Usmg an isolated perfused hver system Eyer and co-workers (Eyer et al 1980) demonstrated that hver also formed N-oxidized metabohtes of andme, and that the terminally oxidized metabohte nltrosobenzene was capable of forming covalent adducts with liver protein The N-oxidation pathway was judged to be inconsequential for hver, however, in that addmonal experiments showed that liver rapidly reduced N-oxidized aniline metabolites back to the parent compound Thus, in hver the predominant metabolic pathways revolve nng hydroxylatlon acetylation, and other assocmted conjugation reactions (F~g 3) The metabolic handhng of N-oxl&zed aniline metabohtes by erythrocytes contrasts sharply with that of liver Further isolated perfused liver experiments indicated that small amounts of phenylhydroxylamine apparently escape the liver and are taken up by erythrocytes Once within the erythrocyte, phenylhydroxylamlne is rapidly oxi&zed to mtrosobenzene by oxyhaemoglobm with concurrent formation of methaemoglobin (Eyer et al 1980) Although the erythrocyte, hke hver, has the capacity to reduce nltrosobenzene back to phenylhydroxylamine and anlhne, In the presence of oxyhaemoglobln reoxidatlon to nltrosobenzene predominates (Fig 3, Eyer & Lierhelmer, 1980) In fact, the abdlty of the erythrocyte to cycle N-oxl&zed aniline metabohtes in this manner accounts for their potent ability not only to produce methaemoglobinaemia but also to gener- ' ate metabohtes capable of forming covalent adducts with erythrocyte proteins (Eyer & Lierhelmer, 1980, Kiese & Taeger, 1976) Evaluation of the disposition of ~4C-anillne in rodents has provided additional support for the central role of the erythrocyte m the spleen-directed toxicity of aniline In rats administered single oral doses equivalent to the dally doses used in the CIIT (1982) bloassay, radioactivity was uniformly &stnbuted and cleared from several organs, including the hver and spleen (Fig 4) However, radioactivity was selectively retained in the erythrocyte relatwe to plasma or other organs examined (Bus et al 1978) Addmonal studies indicated that ra&oactlvity retamed in erythrocytes was covalently bound When multiple doses of radlolabelled amhne were administered to rats, accumulation of covalently bound radioactivity was then observed in the spleen (Fig 5) No current accumulation of covalently bound radioactwity was seen in a nontarget tissue, the hver (Bus & Sun, 1979, Sun & Bus, 1980) Since amhne is acutely toxic to erythrocytes (Kaese, 1974) and chronic exposure produces evidence of red-blood-cell damage (increased retIculocyte count and Heinz bodies, decreased haematocrit, hemoglobin and erythrocyte counts, CIIT, 1982), splemc accumulation of radioactivity may be due to scavenging of damaged erythrocytes carrying anlhne-denved material A recent study characterizing the dose-dependent accumulation of covalently bound ra&oactlvlty m the

100

0

50

0

623

too E >

50

I

1o 05

0 1

I 1

I 2

I 4

I ,6 Time

I 8 after

I 12

I 24

dosing ( h r )

Fig 4 Disposition of radioactivity denved from t4C-anihne HC1 in erythrocytes (A), liver (@), plasma (@) and spleen (O) in rats treated with a single oral dose of 100 mg aniline HC1/kg body weight expressed as #g-eqmvalents of 14C-aniline HC1/ml of plasma or packed erythrocytes or/g wet weight of tissue (Bus et al 1978, Bus & Sun, 1979) spleen provided data consistent with the hypothesis outlined in the previous paragraph (Robertson et al 1983). Male rats given five daily doses of 1 to 100mg/kg body weight of 14C-anillne accumulated covalently bound radioactivity in red blood cells and liver m a hnear dose-dependent manner (the amount of bound material accumulating in liver was slight) However, accumulation of radioactivity in the spleen was seen only at dally doses greater than 10 mg/kg body weight and always was associated with measurable increases in spleen weight (indicative of splenic scavenging of damaged erythrocytes) These results suggested that anihne-denved radloacUvlty did not selectively accumulate in the spleen at doses of andlne insufficient to cause red-blood-cell toxloty It is interesting to note that m the mouse, which is resistant to splenic toxicity induced by amhne and structurally-related compounds, specific retention of ~4C-amhne-derlved was not observed m erythrocytes (Robertson et al 1983) Furthermore, radioactwity &d not accumulate m mouse spleen after multiple doses of 14C-amllne, nor was any splemc enlargement observed The covalent binding data in conjunction with the pathology data from chromc stuches and the available genotoxlclty results suggests several mechanistic possibilities for the splemc injury and earclnogemoty of amline and its structurally related compounds (Fig 6) First, the accumulation of racholabel vathm the spleen may result from splemc scavenging of andlne-damaged red blood cells containing free and bound amline and/or metabohtes The scavenging of erythrocytes by the spleen may produce localized elevated concentrations of toxic material

J S Bus and J A POPP

624 500

T 400

T

c a. 300 > e I

3

200

T 100

L,ver SpLeen BLood

L,ver

SpLeen BLood

L,ver SpLeen BLood

Fig 5 Covalently bound radioactivity m hver, spleen and blood of male rats administered ~4C-amhne for 1, 3 or 10 days (I00 mg/kg/day, orally) expressed as # g-eqmvalents of bound t4C-amhne HCl/mg protein Ra&oactLwty was determined 24 hr after the last amhne dose (Bus & Sun, 1979, Sun & Bus, 1980) Mixed function oxtdases i,-

Liver AniLine

~onjugat~ed

Excretion .,i

I

products

AniLine

Excretion.,,=

Oxidized metaboLites

~=

PhenyLhydroxyLamine Oxy- Hb ~ ,

Detoxifled metoboL Ires

Met_HbP l'~ Nltrosobenzene

Erythrocyte

CovaLenIty bound products

Toxicity

SpLenic scavenging of damaged erythrocytes

1 DeLivery of aniline and/or toxic metoboLItes from erythrocytes

2 Deposition of erythrocytes and ceLLuLar debris

Va2Lor

Iron congestion accumulation ~_ DNA ~" , o .aemorrnag. . . . . . . . .

sC~:nalLcent ab~rndmogL; ° uLes

±,ox,o,,, SpLeen

~

~"

/

Tumours

Fig 6 Proposed mechamsuc scheme for the spleen-&rected toxicity of anlhne and structurally-related compounds

Splenic toxicity of amhne compounds within the spleen, resulting m dose-dependent binding of metabohte(s) to critical macromolecules in the spleen In this regard, o-amlnophenol, which was the only s~gmficant metabohte of andme causing slgnlficantformatlonofSCE(Wdmeretal 1981), has been reported to be metabohzed by mammalian systems to tughly reactive o-qumommme mtermedmtes capable of reacting with cellular macromolecules A study evaluating the disposition of mtrobenzene, however, suggests that anlhne-denved material in the spleen may not be bound to splenic macromolecules (Goldsteln & Rlckert, 1984) Nltrobenzene, which Is extensively reduced by intestinal mlcroflora to amhne, was distributed In rats in a pattern virtually ldenucal to that of aniline Chromatographic analysis of the ra&oactwlty &stnbuted to the spleen revealed that the majority of the bound isotope was associated with haemoglobin, or possible proteolytlc products of haemoglobin These findings suggest that the accumulation of covalently bound label in the spleen after anlhne dosing was hkely to be due to deposition of radiolabei from scavenged erythrocytes, and not to any specific binding to critical splenic macromolecules A recent study (McCarthy et al 1985) has directly characterized the binding of andmeodenved radioactivity to DNA, R N A and protein m a number of organs of rats, including the spleen In this study, animals were dosed with nonradioactive aniline for 7 days followed by radiolabelled aniline on the eighth day Increased bmdmg of radlolabel in the spleen was noted in R N A and protein, but not DNA, when a low single dose of amhne was used (50 mg/kg body weight, lp) At a higher dose (250 mg/kg body weight, lp), binding of radlolabel to D N A was apparent in the kidney, large mtestme and spleen, the kidney, which is not a target for amhne carcmogemclty, had the greatest level of D N A binding (covalent binding index of 14 2 compared with 3 7 In the spleen) The covalent binding m&ces obtained, however, were very much lower than those obtained with other carcinogenic agents such as aflatoxm B~ (17,000) and 2-acetylammofluorene (560) Unfortunately, this study &d not address the source of the macromolecules in the spleen that contained the covalently bound ra&oactwlty In view of the abdlty of the spleen to scavenge damaged erythrocytes containing covalently bound material, it is possible that the labelled macromolecules in the spleen were of erythrocytlc ongm If this ~s the case, these data would not support a &rect-actlng genotox~c mechamsm of splemc carcmogemcity The reaction of aniline metabohtes with splemc macromolecules ~s not the only mechanism, however, by which potentml genotoxlc injury could be lnmated in the spleen The observation that both chromc and acute administration of amhne results in marked splenic enlargement suggests an alternative, or perhaps conjunctive, mechamsm of tOXlC~ty. Scavengmg of erythrocytes leads not only to splemc enlargement and hyperplasla, but also to significant haemoslderm deposmon, indicative of iron overload Iron overload in liver (Jacobs, 1980) and spleen (Heys & Dormandy, 1981) has been assocaated with Ussue damage possibly mediated by lron-catalysed free-radical

625

reacUons Such reacUons result in a variety of tissuedamaging reactions including hpld peroxldatlon, protern degradation and D N A strand breaks (Bus & Gibson, 1984; Halhwell & Gutteridge, 1986, Weir, Gibson & Peters, 1984) Thus, the spectrum of toxic effects of anlhne m the spleen may not be due to an interaction of anlhne or its metabohtes w~th splenic macromolecules, but rather may reflect the rateraction of physxologlcal compensatory and repair mechanisms (increased spleen size and cell hyperplasla) and ongoing free-radical reactions associated with chronic deposmon of erythrocytlc debris in the spleen (Fig 6) Significance Exploration of the mechanism of toxicity of andme and related compounds has provided reformation important in determining the potential risk associated with exposure of humans to these compounds If the splemc tumongemclty of these agents is linked to an mmal toxic event m the erythrocyte, It appears that splenic carcmogemclty may be determined by a defnable threshold dose of the compound 0 e the events leadmg to carcmogemcity are not set into motion untd the abdlty of the red blood cell to cope with the toxic result is exceeded) Indeed, the dose response curve of the tumorlgemoty of aniline (NCI, 1978, CIIT, 1982) and the pattern of accumulation of bound radiolabel derived from aniline in the spleen (Robertson et al 1983) suggest that such a threshold may exist REFERENCES Abe S & Sasakl M (1977) Chromosome aberrations m sister chromatld exchanges m Chinese hamster cell exposed to various chemicals J natn Cancer Inst 58, 1635 Bus J S (1983) Aniline and mtrobenzene erythrocyte and spleen toxicity CIIT Acuvmes 3 (No 13), 1 Bus J S & Gibson ] E (1984) Role of activated oxygen in chemical toxicity In Drug Metabohsm and Drug Toxlczty Edited by J R Mitchell & M G Hornmg p 21 Raven Press, New York Bus J S, RJckert D E, Norton R M & Gibson J E (1978) The pharmacokmetlcs and metabolism of aniline hydrochlonde m Fischer 344 rats Toxic appl Pharmac 45, 256 Bus J S & Sun J D (1979) Accumulation of covalent binding of radioactivity in rat spleen after ~4C-anlhneHC1 administration Pharmacologist 21, 221 Chemical Industry Institute of Toxicology (1982) 104-Week Chronic Toxicity m Rats Aniline Hydrochlonde Final Reports Vol I and II Eyer P, Kampffmeyer H, Malster H & Rosch-Oehme E (1980) Biotransformatton of mtrosobenzene, phenylhydroxylamme, and aniline m the isolated perfused liver Xenobtoma 10, 499 Eyer P & Lierhelmer E (1980) Blotransformatlon of mtrosobenzene m the red cell and the role of glutathlone Xenobtotwa 10, 517 Goldstem R S & RIckert D E (1984) Macromolecular covalent binding of [14C]-mtrobenzene In the erythrocyte and spleen of rats and mice Chemtco-Bzol Interactions 50, 27 Goodman D G , Ward J M & Retchardt W D (1984) Splenic fibrosis and sarcomas m F344 rats fed &ets containing aniline hydrochlonde, p-chloroamhne, azobenzene, o-tolmdme hydrochlonde, 4,4'-sulphonyl-

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dmndme, or D & C Red No 9 J natn Cancer Inst 73, 265 Halhwell B & Guttendge J M C (1986) Iron and free ra&cal reacUons two aspects of anttox~dant protection Trends btol Sct 11, 372 Heys A D & Dormandy T L (1981) Lipid peroxldaUon m iron-overloaded spleens Chn Sct 60, 295 Jacobs A (1980) The pathology of iron overload In Iron m Btochemzstry and Medicine II E&ted by A Jacobs & M Worwood p 247 Academic Press, London Kao J , Faulkner J & Bridges J W (1978) Metabohsm of anlhne m rats, pigs and sheep Drug Metab Dtspos 6, 549 Klese M (1974) Methemoglobmemza A Comprehenswe Treattse CRC Press, Cleveland, OH Klese M & Taeger K (1976) The fate of phenylhydroxylamme m human red cells NaunynSchmtedeberg's Archs Pharmac 292, 59 McCarthyD J,WaudW R,StruckR F &HdlD L (1985) Dlsposmon of metabohsm of amhne m Fischer 344 rats and C57BL/6 x C3H F~ mice Cancer Res 45, 174 National Cancer Institute (1977) Bloassay of Dapsone for Possible Carcmogemoty Tech Rep Ser No 20 U S Govt Print Off, Washington, DC National Cancer Institute (1978) B~oassay of Andme Hydroxychlonde for Possible Carcmogemclty Tech Rep Ser No 130 U S Govt Print Off, Washington, DC Nahonal Cancer Institute (1979a) Bloassay of pChloroamhne for Possible Carcmogemclty Tech Rep Ser No 189 U S Govt Pnnt Off, Washington, DC National Cancer Institute (1979b) Bloassay of Azobenzene for Possible Carcmogemclty Tech Rep Ser No 154 U S Govt Print Off, Washington, DC Nahonal Cancer Institute (1979c) Btoassay of o-Tolmdme Hydrochlonde for Possible Carcmogenlc~ty Tech Rep Ser No 153 U S Govt Print Off, Washington, DC National Cancer Institute (1982) Carcinogenesis B~oassay o f D & C Red No 9 m F344 Rats and B6C3F1 Mice (Feed Study) Tech Rep Ser No 225 U S Govt Print Off, Washington, DC Parke D V (1980) The metabohsm of [14C]amlme m the rabbit and other ammals Bzochem J 77, 493 Parod~ S, Pala M , Russo P , Zumno A , Balbl C , Albml

A , Valeno F , Clmberle M R & Santl L (1982) DNA damage m hver, kidney, bone marrow, and spleen of rats and rmce treated with commercial and purified amhne as determined by alkahne elutlon assay and sister chromatld exchanged reduction Cancer Res 42, 2277 Paro& S, Zumno A , Ottagglo L , DeFerran M & Santl L (1983) Lack of correlation between the capabdRy of inducing s~ster-chromat~d exchanges m wvo and carcmogemc potency, for 16 aromatic amme and azo derivatives Mutanon Res 108, 225 Peters J H , Gordon G R , Tanaka W & Semmon V F (1978) Mutagenesls of dapsone and its denvatwes m Salmonella typhlmunum Fedn Proc Fedn Am Socs exp Btol 37, 450 Prasad I (1970) Mutagemc effects of the herbicide 3',4'-dlchloroproprloamhde and Rs degradation products Can J Mtcrobzol 16, 369 Robertson P Jr, Cox M G & Bus J S (1983) Response of the erythrocyte and spleen to amhne insult m Fischer 344 rats Toxtcologlst 3, 128 Rosenkranz H S & Pomer L A (1979) Evaluation of the mutagemclty and DNA-modffymg actlwty of carcinogens and noncarcmogens m mlcrobml systems J natn Cancer Inst 62, 873 Smith J N & Wflhams R T (1949) Stu&es m detoxlcatlon 23 The fate of amhne m the rabbR. B;ochem J 44, 242 Sun J D & Bus J S (1980) Companson of covalent binding of 14C-aniline HC1 m red blood cells, spleen and hver of rats Pharmacologtst 22, 247 Swenberg J A , Petzold G L & Harbach P R (1976) In vttro DNA damage/alkahne elutlon assay for predicting carcmogemc potentml Btochem blophys Res Commun 72, 732 Wemberger M A , Albert R H & Montgomery S B (1985) SplenotoxlcRy assocmted with splemc sarcomas m rats fed high doses of D & C Red No 9 or amhne hydrochlonde J natn Cancer Inst 75, 681 Weir M P , Gibson J F & Peters T J (1984) Haemosldenn and tissue damage Cell B, ochem Functton 2) 1986 Wdmer J L , Khgerman A D & Erexson G L (1981) Sister chromahd exchange reduction and cell cycle lnh~bmon by andme and ~ts metabohtes m human fibroblasts Envtr Mutagen 3, 627