SYMPOSIUM Cticrnical Carcinogencsis: Xcnobiotics anti Uiotransforrnation

TOXICOLOGIC PATIIOLOCY Vol. 12, No. 1, 1984

Thyroid and Chemical Hepatocarcinogenesis: Further Insights

from the Hepatocarcinogen Zami 1305.* MARCO PRESTA, TERESA ZAVANELLA', CECILIA MAZZOCCIII, SILVIA ZILIANI,

GIOVANNA MAZZOLENI, DANIELA CALOVINI, MAKINA BRAGA AND GIOVANNI RAGNOTTI

Department of Basic Biomedical Sciences, Chair of General Pathology, Faculty of Medicine, University of Brescia, 25224 Brescia, Italy, 'Institute of Zoology, Faculty of Sciences, University of Milan, 20233 Milan, Italy

ABSTRACT The 0-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-ter~-butylaminopropan-2-

01 (ZAMI 1305), oncogenic to the liver of the female but not of the male Wistar rat, was used to investigate some aspects of the relationship between liver and thyroid during chemical hepatocarcinogenesis. Thyroidectomy (TDX) strongly reduces the amount of hepatic DNA damage induced by a single administration of ZAMl 1305 in the female Wistar rat. One week of treatment with triiodothy- ronine (T,) completely restores the susceptibility of the liver of thyroidectomized animals to the genotoxic activity of the molecule. The amount of hepatic DNA damage in intact females varies with the age of the animal, being maximal in rats of 4-8 weeks of age, when T3 serum concentration are also maximal. An increase of relative thyroid weight, coupled with histological hyperplasia of the gland, is observed in female Wistar rats treated for 6 months with ZAMl 1305. Minimal changes of the thyroid are observed in ZAMl 1305-treated male rats. The increase of relative thyroid weight in female rats appears to be related to the severity of preneoplastic and neoplastic liver changes. These findings and several suggestions from the literature lead us to propose a model for the interaction between liver and thyroid during chemical hepatocarcinogenesis.

I NTKODUCTION

In 1948 Paschkis et al. (1) demonstrated the protective effect of thiouracil on the induc- tion of liver tumors by 2-acetylaminofluorene (2-AAF). Subsequently, various authors re- ported on the inhibition of hepatocarcinqje- nesis in hypothyroid rats (2-6). Thyroid actiu- ity was also shown to modulate the rate of growth of transplantable hepatic tumors both in vitro (7) and in vivo (8-12). Conversely, modifications of serum levels of thyroid hor-

Prescntcd at the Second International Symposium Spon- sored by the Universities of Sassari and Cagliari, Session I: 'Genetic exprcssion and Cell Transformation". October 12- 15. Alghero. Italy. This Symposium section will be continued in \'olumc 12. Number 2. 1981.

mones and of the histological appearance of thyroid were reported to occur during hepa- tocarcinogenesis (1 3-14). These findings seem therefore to suggest the existence of a mutual influence between liver and thyroid during induction and growth of hepatic tu- mors.

The present report describes our observa- tions on the interplay between liver and thy- roid during chemical hepatocarcinogenesis induced by the compound DL-1-(2-nitro-3- methyl-phenoxy)-3-tert-butylamino-propan- 2-01 (ZAMI 1305) (Fig. I), whose oncogenicity have been recently demonstrated in our lab- oratory (15). This compound, which belongs to the class of P-adrenoreceptor blocking

49

50 PRES'I'A ET AI,. TOXICOI.OGIC PATHOLOGY

ZAMI 1305 ZAMl 1327

FIG. 1-Chemical structure of ZAMI 1305 and of ZAMl 1327.

TABLE I-Pharmacologic Activities of DL-ZAMI 1305, DL-ZAMI 1327 and DL-propranolol"

Local An- &blocking Activity aesthetic

DKUG Activity

( d k n bodv wt) (mn/ml) ED,,, EDSO

DL-propranolol 64 (1 .O) 1.4 (1.0) DL-ZAMI 1305 6 (10.7) 0.7 (2.0) DL-ZAMI 1327 7 (9.1) 2.5 (0.6)

-' p-blocking activity was evaluated as antagonism to iso- prcnalinc-induced tachycardia in the anesthetized rat (45) and local anaesthetic activity as suppression of the cornea reflex in the guinea pig (46). EDso represents the dose giving the 50°/0 effect. In parenthesis is the potency with respect to DL-propranolol.

agents (Table I), induces the growth of carci- nomas in the liver of the female but not of the male M'istar rat (15). An isomer of ZAMI 1305, DL-1-(2-nitro-5-met hyl-phenoxy)-3- tert-butylaminopropan-2-01 (ZAMI 1327), though having similar chemical structure (Fig. 1) and pharmacologic activity (Table I), is without effect on the liver of rats of both sexes (15); i t has therefore been utilized as a negative control for ZAMI 1305.

RESULTS AND DISCUSSION 1) Role of thyroid activity on the liver DNA

darnaging capacity ofZAMI 1305. The induc- tion of liver tumors in rats fed 2-AAF is partially inhibited by a concurrent treatment with the goitrogen thiouracil (1, 2). The in- hibition of liver carcinogenesis by 2-A4F in surgically thyroidectomized animals co'rifjrm the anticarcinogen effect exerted by chrohic hypothyroidism (4-6). Administration of thy- roid powder to thyroidectomized animals re- stores the oncogenicity of 2-AAF (5).

These data, which underline the relevant role of thyroid activity in hepatocarcinoge- nesis, raise the question of which stage in carcinogenesis is inhibited by thyroid defi- ciency. In 1953, Bielschowsky and Hal1 (3) demonstrated that thyroidectomy, performed

prior to the administration of the carcinogen, prevented the development of liver neo- plasms, while thyroidectomy performed after the administration of the carcinogen did not affect neoplastic transformation and tumor growth. These findings were confirnied by Goodall (6), who demonstrated that in thy- roidectomized animals the treatment with thyroid digest after aminofluorene adminis- tration, did not restore susceptibility to the carcinogen. These data, therefore, suggest that thyroid activity influences liver carci- nogenesis at or before the stage of initiation. This hypothesis is supported by the fact that thyroid hormones have been demonstrated to be iiecessary for the in vitro neoplastic transformation by X-rays of mouse fibroblasts and hamster embryo cells (16, 17).

The interaction between chemical carcin- ogens and cellular DNA is considered to be a crucial event in the induction of neoplastic transformation (18, 19). This interaction may result in DNA damage, demonstrable as al- kaline labile sites by alkaline sucrose gra- dient analysis (20-22). In this regard onco- genic ZAMI 1305, but not the non-oncogenic ZAMI 1327, induces liver DNA damage when administered to the female Wistar rat (23).

To clarify the role of thyroid activity on initiation by chemical hepa tocarcinogens, we have compared the DNA damaging effect ofZAMIl305 in intact, thyroidectomized and in thyroidectomized triiodothyronine (TJ- treated female M'istar rats. As shown in Fig. 2, thyroidectomy (TDX) strongly reduces the amount of hepatic DNA darnage induced by ZAMI 1305; conversely, T,-administration to thyroidectomized animals completely re- stores the DNA damaging effect of the mole- cule. Serum levels of thyroid hormones have been shown to vary during the life span of the rat (24). Thus, we have also evaluated the effect of ZAMI 1305-induced liver DNA dam- age in female M'istar rats of different ages (Fig. 3). The amount of DNA damage appears to be related to the serum levels of thyroid hormones,"being higher in rats of 4-8 weeks

Vol. 12, No. 1 , 1984 THY R 0 ID AND C HEM I C A L 1-1 E PAT0 C A R C I N 0 GENES IS 51

INTACT a 1 I

TOX TOX+T3

FIG. 2-Effect of thyroidectomy (TDX) and T3- treatment on hepatic DNA damage induced by ZAMl 1305 in female Wistar rats. DNA damage was evaluated by alkaline sucrose gradient analysis 14 hours after i.p. injection of ZAMl 1305 (75 mg/ kg body wt) (23). TDX was performed 10 days before experimentation and T3 was administered S.C. at 5 c(g/lOO g body wt./day for 1 week before ZAMl 1305 administration. DNA damage is ex- pressed as DE/Dc ratio (23). The value and varia- bility of DE/Dc in untreated control animals is shown as a bracket. Each bar is the mean C S.E.M. of 3-11 animals.

t - 2.0. u

\ W

0

n

- a

1.5. 5

0

1-3 4-8 10-30 A G E (weeks)

FIG. 3-Age-related hepatic DNA damage in- duced by ZAMl 1305 in the female Wistar rat. DNA damage was evaluated by alkaline sucrose gradient analysis (23) 14 hours after ZAMl 1305 i.p. administration (75 mg/kg body wt.). The amount of DNA damage is expressed as DE/Dc ratio (23). Each bar is the mean 2 S.E.M. of 5-13 animals. The values of serum concentrations of T3 are calculated from literature data (24).

of age, when the concentration of ‘I’, and T, in serum is also maximal (24).

These findings, therefore, support the hy- pothesis that thyroid hormones are involved in liver initiation by chemical carcinogens. These hormones could modulate the geno- toxic activity of chemical carcinogens through an endocrine-dependent effect on the activity of the microsomal mixed func- tion oxidase system (25-27), and/or by stim-

. .

ulating cellular replication (28, 29). Thyroid hormones may, therefore, indirectly influ- ence the formation of electrophilic carcino- genic metabolites able to bind to the target macromolecules (18, 19), and/or they may modify the susceptibility of DNA to the action of the carcinogen by stimulating liver DNA synthesis. Relevant to the latter possibility’is the well documented greater sensitivity of the liver to chemical carcinogens during DNA replication (30-34).

Even though the data obtained in our lab- oratories and in other (3, 6) suggest that thy- roid activity has a relevant role in the initia- tion phase of hepatocarcinogenesis, the possibility that thyroid hormones are also involved in liver tumor promotion and growth cannot be excluded. It is, in fact, well documented that liver regeneration (28) after partial hepatectomy and the rate of growth in vitro and in vivo of transplantable hepa- tomas (7-12) depends on thyroid activity. Moreover, thyroid digest increases the num- ber and the rate of growth of liver tumors in pituitary dwarf mice treated with aminoflu- orene (35). It must be pointed out, however, that no modification of liver tumor growth is observed in rats thyroidectomized 13 weeks after aminofluorene feeding (35).

2) Thyroid modifications and hepatocarcin- ogcnesis by ZAMI 1305. As thyroid activity affects hepatocarcinogenesis by chemical carcinogens, liver neoplasms in turn appear to induce thyroid modifications. Histological modifications of the thyroid gland during chemical hepatocarcinogenesis have, in fact, been reported (13, 14). A histologically hy- peractive thyroid, with a concomitant reduc- tion in T4 and T, serum levels, is observed in female rats treated with 2-AAF. The same treatment in male rats induces, instead, hy- potrophy of the thyroid gland, accompanied by a transient reduction of T, and T4 serum levels. Moreover, changes of serum concen- trations of thyroid hormones have been re- ported to occur in hepatomas-bearing rats (8, 11, 29) and in patients with hepatocellular carcinomas (36).

We decided, therefore, to investigate the histological appearance of the thyroid gland in rats treated for 6 months with different doses of ZAMI 1305 (37). After this period of treatment various types of preneoplastic and neoplastic alterations are present in the liver of female rats, while no histological altera- tions are detectable in the liver of male rats (15). The comparison of the histological ap-

52 PRESTA ET AI,. TOXICOLOGIC PATHOLOGY

pearance of the thyroid in ZAMI 1305-treated female rats compared to that of male rats could, therefore, provide information on changes specifically related to the presence of hepatic tumors (hence detectable only in females). Direct effect of the molecule on the thyroid gland should be detectable in both sexes, even though possible different phar- macologic effects of the molecule in the two sexes cannot be excluded.

ZAMI 1305 administration causes a dose- dependent increase of relative thyroid weight (g wet tissue/g of brain) in female rats; the increase is less pronounced in male rats (Fig. 4). Six months administration to female rats of the nononcogenic &blockers ZAMI 1327 (400 mg/kg body wt/day) or practolol (300 mg/kg body wt/day) results in a slight in- crease (+7% and +2%, respectively) of rela- tive thyroid weight, similar to that observed in ZAMI 1305-treated male rats. These data suggest that the moderate increase of thyroid weight observed in ZAMI 1305-treated male rats and in ZAMI 1327 or practolol-treated female rats is. related to the pharmacologic activity of the molecule. &blocker adminis- tration causes a decrease in the serum con- centration of T3 (38-41). This, by a feedback mechanism, i t could lead to increased activ- ity and size of the thyroid. In ZAMI 1305- treated female rats, the more pronounced increase of thyroid weight may be related, at least in part, to the presence of preneoplastic and neoplastic liver alterations. To confirm this hypothesis, the animals were distributed into 3 groups, according to the type of the

0 2 5 5 0 100 ZAM11305 (mg/ kg body w t )

FIG. 4-Relative thyroid weight in Wistar rats after 6 months chronic treatment wi th ZAMl 1305. Male and female Wistar rats were treated per 0 s for 6 months at the doses indicated. Relative thy- roid weight was calculated as mg wet wt./g brain wt. Each point i s the mean k S.E.M. of 12-18 animals. (01, males. (01, females.

predominant liver lesion (clear cell foci, neo- plastic nodules, hepatocellular carcinomas), but independent of the' dose of .ZAMI 1305 administered. A s shown in Fig. 5, relative thyroid weight is directly related to the se- verity of hepatic lesions.

At histological examination, a dose-de- pendent increase in the incidence of animals showing moderate to marked thyroid hyper- plasia was observed in ZAMI 1305-treated female rats (Fig. 6), while no modifications were detectable in male rats. I t should be noted, hoivever, that when female rats were grouped according to the type of the predom- inant liver lesion the percentage of animals

FIG. 5-Preneoplastic and neoplastic liver le- sions and relative-thyroid weight -in ZAMl 1305- treated female Wistar rats. The animals were treated per 0 s for 6 months with different doses of ZAMl 1305 (see Fig. 4). They were distributed, independently o f the dose administered, according to the type of the predominant liver lesion. Each bar i s the mean k S.E.M. o f 12-19 animals.

ZAMll305 (mg/ kg body w t / d r y )

FIG. 6-Thyroid hyperplasia in Wistar rats treated with ZAMl 1305 for 6 months. Thyroid hyperplasia i s defined by the presence o f tall and columnar cells and the reduction of the follicular size (37). Each value represents the percentage calculated on groups of 12-18 rats. (O), males; (0), females.

Vol. 12, No. 1,1984 THYROID AND CHEMICAL HEPATOCARCINOGENESIS 53

showing thyroid hyperplasia was not related to the histological appearance of the liver (Fig. 7).

These findings indicate that thyroid alter- ations, consisting of an increase in tissue mass

FIG. 7-Thyroid hyperplasia in female Wistar rats bearing hepatic preneoplastic and neoplastic ZAMl 1305-induced alterations. The animals were treated for 6 months with different doses of ZAMl 1305 (see Fig. 6). They were distributed, independ- ent of the dose; administered, according to the type of predominant liver lesion. Each bar represents the percentage calculated on groups of 12-18 an- imals.

coupled with hyperplasia, do exist in hepa- tocarcinogen-treated animals and they ap- pear due, at least in part, to the presence of preneoplastic and neoplastic liver alterations and not to a specific toxic effect of the mole- cule on the gland. Even though these altera- tions may suggest either a hypofunctioning or a hyperfunctioning thyroid, the high inci- dence of liver tumors in these animals would exclude, for the reasons discussed above, the hyperfunctional hypothesis. Hyperactivity of the thyroid gland during chemical hepatocar- cinogenesis may be explained by hypothes- izing a progressively higher uptake of thyroid hormones during liver neoplastic progres- sion, as suggested by the increased concen- tration of receptors for T3 in rat and human hepatomas (42, 43). Higher hepatic uptake of thyroid hormones has also been shown to occur during liver regeneration (44), suggest- ing that this phenomenon is a common fea- ture of liver cells during sustained periods of rapid replication. Moreover, in patients with hepatocellular carcinomas (36), the trans- formed liver seems to synthesize a greater quantity of thyroxine-binding globulin (TBG). This, coupled to a decrease of free T3 serum concentration, may result, by a feed-

I N I l l AT I0 N PROMOTION and PROGRESSION

1 ---- THYROID

HYPERPLASIA I I I I I I I I

t t s e r u m

1 t l3 ,14 release

THYROIO

I I I I I I I I

hepatocytes multiplication

TBG synthesis I T 3 l iver uptake

modulation of hepatic drug metabolism t

PRENEOPL ASTIC

LIVER

r-----i - - - - - - - - - I I V

INITIATED

carcino j en

FIG. 8-Mutual relationship between thyroid and liver during chemical hepatocarcinogenesis. (-): proved; (--- -): postulated; (I), increased; (J), decreased.

54 PRESTA ET AL. TOXICOLOGIC PATHOLOGY

back mechanism, in an increase of TSH levels, with a consequent stimulation of thy- roid activity.

From data in the literature and from our observations on i) the role of thyroid hor- mones on the DNA damaging capacity of ZAMI 1305, and ii) thyroid alterations during hepatocarcinogenesis by ZAMI 1305, it seems possible to hypothesize the following recip- rocal influences (Fig. 8) between thyroid and liver during chemical hepatocarcinogenesis:

1) thyroid hormones, stimulating hepatic cellular multiplication and affecting drug me- tabolism, “facilitate” initiation by chemical hepatocarcinogens; 2) the anabolic activity of thyroid hormones allows tumor progression and growth, as shown for the thyroid hor- mone-dependent growth of several hepato- mas; 3) preneoplastic and neoplastic liver stimulates thyroid activity shown by greater uptake of thyroid hormones and an elevated production of thyroxine-binding globulin; and 4) the elevated thyroid activity may, in turn, stimulate tumor growth establishing a vicious, self-sustaining cycle.

Further studies are necessary to confirm this hypothesis. Hepatocarcinogenesis by ZAMI 1305 represents a suitable model for additional studies on the mutual influence of liver neoplastic transformation and thyroid activity.

ACKNOWLEDGEMENT This work was in part supported by grants

from the C.N.R. (82.02818.04) and from the Minister0 dclla Pubblica Istruzione (M.P.I., 40%, National Project of ExperimentaI On- cology).

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