TCDD resistance is inherited as an autosomal dominant trait in the rat

Toxicology Letlers, 50 (1990) 49-56

49

Elsevier

TOXLET

02232

TCDD resistance is inherited as an autosomal dominant trait in the rat

Raimo Pohjanvirta Department of Environmental Hygiene and Toxicology, National Public Health Institute, Kuopio (Finland) (Received

31 May 1989)

(Accepted

21 July 1989)

Key words: 2,3,7,8-Tetrachlorodibenzo-p-dioxin;

TCDD;

Rat strains;

Inheritance

of sensitivity

SUMMARY In the present

study,

genetic

Evans)

and the most

TCDD

as the Han/Wistar

progeny

crossings

TCDD-resistant

the distribution

were performed (Han/Wistar)

rats irrespective of resistant

and susceptible

3) autosomal

genes displaying

tive co-effect.

These data show that, in contrast

the dominant

trait in the rat. Moreover, of TCDD

the most TCDD-susceptible The Fl offspring

of the sex of their Han/Wistar

by 2 (possibly

exclusive determinant

between

rat strains. phenotypes

complete

was consistent

dominance,

to earlier findings

the results challenge

parents.

independent

In test-cross

with inheritance segregation,

in mice, TCDD

the current

(Long-

were as resistant

resistance

to

and F2 regulated

and an addiseems to be

view that the Ah-locus

is the

sensitivity.

INTRODUCTION

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely toxic world-wide pollutant, whose mode of action has remained elusive. A single dose of TCDD in the ,ug/kg range causes a host of biochemical and pathological effects which depend on species to an appreciable extent [l]. However, in mice a number of these effects (such as induction of hepatic cytochrome P-450 associated enzyme activities, immunosuppression accompanied by thymic atrophy, cleft palate formation, hepatic porphyria, myelosuppression, and lipid peroxidation) have been shown to segregate with a single genetic locus, the Ah-locus [2-91. The Ah-locus is part of the murine Ahcomplex, a gene battery comprising at least 10 closely related genes [lo]. The major regulatory gene product of the Ah-complex is a receptor molecule specific for TCDD, Address for correspondence:

R. Pohjanvirta,

Department

tional Public Health

Institute,

P.O.B. 95, SF-70701

0378-4274/90/$3.50

@ 1990 Elsevier Science Publishers

of Environmental

Kuopio,

Hygiene

Finland.

B.V. (Biomedical

Division)

and Toxicology,

Na-

50

encoded by the Ah-locus [l I]. This receptor controls the expression of related structural genes [ 12,131. Ah-responsiveness (as determined by enzyme induction) is usually inherited as a simple autosomal dominant trait [1416]. Responsive inbred strains of mice (typified by C57BL/6) have a similar number of functional Ah-receptors to that of other species, while non-responsive strains (typified by DBA/2) are devoid of detectable receptors [ 17,181. In addition to the above-mentioned individual end-points of toxicity, the Ah-locus also segregates with TCDD lethality in mice [19,20]. The only manifestation of TCDD toxicity where alleles other than those of the Ah-locus have been implicated is the murine skin lesion (hyperkeratosis, epidermal hyperplasia, and sebaceous gland metaplasia), which requires the concurrent contribution of another genetic locus, designated hr [21]. The universally dominant role of Ah-receptors as determinants of TCDD toxicity in general and lethality in particular has recently been questioned [22]. Of special importance in this regard was the finding that Han/Wistar (H/W), the most TCDDresistant (LD50 > 3000 pug/kg) rat strain, and Long-Evans (L-E), the most TCDD-susceptible (LDse ca. 10 pg/kg) strain, have similar levels of functional hepatic Ah-receptors [23]. Therefore, at least in the rat, susceptibility to TCDD lethality might be governed by quite different loci from that operating in the mouse. The present study set out to elucidate the genetic background of TCDD sensitivity in these two strains of rats. MATERIALS AND METHODS

H/W rats (outbred SPF animals) were purchased from the National Laboratory Animal Centre, Kuopio, Finland and L-E rats (random-bred, conventionally raised) from the University of Oulu, Oulu, Finland. For production of the Fl generation, two separate crossings were made. In the first of these, the parent animals consisted of 5 female L-E and 2 male H/W rats (offspring: LEHW); in the second, 8 female H/W rats were mated with 4 male L-E rats (HWLE). A test-cross was obtained by mating 7 L-E females with 3 LEHW males (L-E x LEHW). Finally, the F2 progeny was produced by mating 16 HWLE females with 8 HWLE males (HWLE x HWLE). The rats were allowed to acclimatize to the environment for 5-6 weeks before the onset of the experiments. During the experimental period the rats were housed singly in stainless-steel wire-mesh cages (the LEHW and some of the L-E x LEHW rats were kept in plastic metabolic cages for the first 3 weeks after TCDD exposure to enable measurement of their feed and water intakes). The ambient temperature in the animal room was 21 f 1°C humidity 55 f lo%;, and with a 12 h dark/l2 h light cycle. Only young male adult animals were used in the trials (except for two 25-week-old individuals, all the rats were at age 10-l 3 weeks at onset, the mean weights being 245-290 g). TCDD ( > 99 % pure as determined by GC-MS) was dissolved in corn oil in such a manner that all rats could be given an equal volume (5 ml/kg), as described

51

previously [24]. TCDD was administered as a single intraperitoneal injection on day 0. Three to 5 animals of each generation served as controls and were given corn oil. The rats were weighed twice a week (the rats in metabolic cages every 2 days) and monitored for 49 days. A dose as low as 20 pg/kg of TCDD is usually lethal to all L-E rats while in H/W rats only incidental deaths occur at doses up to 3000 pg/kg [23]. A pilot test was carried out in a small group (4) of LEHW rats. Neither of the TCDD doses tested (50 and 500 pg/kg) proved lethal over a 63-day period, suggesting that TCDD resistance is dominantly inherited. Hence, both Fl offspring were tested at the highest TCDD dose levels attainable with the vehicle used (1000-3000 pg/kg). On the other hand, the doses for the test-cross and F2 generation were selected so as to differentiate both the H/W-like (1000 pg/kg) and L-E-like (20 pg/kg) phenotypes from intermediates. The data were statistically analyzed by two-tailed binomial and chi-square onesample tests [25] with the aid of an SPSS”-programmed VAX 1l/780 computer. RESULTS

The survival rate in the Fl generation was not significantly different from that in H/W rats treated with equal doses of TCDD, irrespective of the sex of parent H/W animals [Table I; the slightly greater mortality in the Fl progeny may be due to poorer health status at the onset of the tests, since necropsy revealed pneumonic lesions in one of the succumbed rats (given 1000 ,ug/kg TCDD) and another rat (not TABLE TCDD

I. LETHALITY

Offspring

TO FI OFFSPRING

TCDD

dose

Survivors/

HWLE

H/W”

a Probability b From

Pohjanvirta

expected

1000

315

2000

415

3000

415

1000

417

2000 3000

617 617

1000

415

2000

415

3000

515

for the rats representing

tance (two-tailed

binomial

the same population

test). For statistical

analysis,

et al. [23]. These data are presented

ratio of survivors

RATS

P-Valuea

exposed

@g/kg) LEHW

OF L-E AND H/W PARENT

and non-survivors.

0.258

0.277

as their H/W parents

in terms of TCDD

resis-

the results at the 3 dose levels were combined. for comparison

and were used in calculating

the

52

used in the experiments) showed signs of respiratory infection]. A further similarity [26] was seen in the pattern of feed intake changes (measured in LEHW rats): an initial steep depression during the first 6 days was followed by a swift recovery to almost control levels as calculated per metabolic body weight (data not shown). In addition, all but 2 of the exposed rats underwent a transient (l-6 days) complete refusal of feed. This behavior is uncommon among TCDD-treated animals in general, but typical of H/W rats exposed to high doses of TCDD [24]. Also similar to H/W rats [26], the Fl progeny did not exhibit any dose-dependence at the doses tested. The outcome in the Fl generation showed that TCDD resistance was inherited as an autosomal dominant trait, the H/W strain being homozygous dominant and the L-E strain homozygous recessive. To provide further insight into the mode of inheritance, a test-cross and F2 generation were produced and their TCDD susceptibility tested (Table II). The data proved fairly convincingly that the number of contributing genes is neither 1 nor 4 (or more). Although the P-values were in no instance below 0.05 for 3 genes, the almost significant figure in the test-cross at 20 ,ug/kg of TCDD (P= 0.055) renders this alternative somewhat unlikely. The best-fitting explanation for all of the results can be provided by assuming that resistance is governed by two additively interacting autosomal loci where the H/W rat is homozygous for dominant and the L-E rat for recessive alleles. TCDD resistance was in no instance associated with coat color (data not shown). DISCUSSION

The information gathered so far of hereditary factors in TCDD toxicity is based solely on studies in inbred mouse strains. These findings have led to the current concept that a single gene encoding the Ah-receptor plays a decisive role in determining TCDD susceptibility such that TCDD sensitivity is the dominant trait. However, the present study clearly demonstrates that this view does not hold in the rat. The strains employed here represent the two extremes of TCDD tolerance in the rat, being over 20-fold more divergent in this respect than their mouse counterparts [19,20]. Nevertheless, they have similar levels of functional hepatic Ah-receptors and respond with a comparable magnitude of enzyme induction to TCDD treatment [23]. The present data reveal that, in contrast to mice, TCDD resistance is the dominant and sensitivity the recessive trait in these rat strains. Furthermore, additive co-operation of two (possibly 3) genes is needed to bring about the phenotypes. It is also of interest to note that neither of the rat strains used in the present study is inbred, whereas all previous mouse studies were performed with inbred strains. The LDso values of other rat strains tested fall between those of the L-E and H/W rat 127-291. It is conceivable that the L-E and the H/W rat are the only strains having either recessive or dominant homozygous alleles for both (or all) of the genes participating in TCDD resistance. Other strains might possess dominant alleles for just one of the genes and thus resemble those rats of the F2 and test-cross offspring in the

53

TABLE TCDD

II LETHALITY

PARENT

TO A GENETIC

TEST-CROSS

AND

F2 GENERATION

OF L-E AND

H/W

RATS

Offspring

TCDD

dose

Survivors/

P-Value”

exposed

@g/kg)

1

2

3

4

6/10

0.754

0.448

0.055

0.005

2118

0.001

0.271

1.000

0.623

0.023

0.127

0.246

0.329

21:21

0.005

0.516

1.000

I .ooo

13121

0.260

0.770

0.110

0.008

0.373

0.373

0.131

0.036

L-E x LEHW (test-cross)

20b 1000 Combined’

HWLE

x HWLE

20

WI 1000 Combined “Probability

of the observed

for the number

outcome

of genes shown

given that the Fl progeny

(two-tailed

tween alleles of the same gene, independent Long-Evans

phenotype

(susceptible

for each of the genes involved, is assumed survivor

to possess

frequencies one-sample

types were calculated

separately

1000 fig/kg the latter phenotype

and HWLE)

co-effect

is assumed

phenotype

(tolerant

were heterozygous

complete

dominance

be-

of the genes. Further,

the

to be homozygous

of TCDD.

the expected

are thus 15/16 (20 @g/kg) and 9/16 (1000 pg/kg).

Both of them survived.

test. The expected

values for the Long-Evans

at both dose levels (assuming from all others),

recessive

to 6 1000 pg/kg of TCDD)

allele for each of these genes. For example,

for 2 genes in the F2 progeny chi-square

and additive

of TCDD)

the Han/Wistar

at least one dominant

(LEHW

test). The model assumes

segregation,

to 3 20 pg/kg

whereas

h Two of these rats received 50 pg/kg c Two-tailed

binomial

and Han~Wistar

that 20 gg/kg differentiates

and the test was performed

pheno-

the former and

on the combined

data.

present study which were susceptible to the dose of 1000 pg/kg, but not to that of 20 Crglkg. The biochemical steps regulated by these resistance genes remain to be determined. Overall, the functional and morphological responses of L-E and H/W rats to TCDD exposure are strikingly similar. The greatest difference we have so far detected occurs in the liver where a typical lesion is only discernible in L-E rats treated with lethal doses of TCDD 1301.Another candidate for the site of action could be the central nervous system, since we have recently shown that the TCDD-induced wasting syndrome can be triggered more readily by intracerebroventricular than peripheral administration in both strains, the response being more severe in L-E rats [31 and un-

54

published data]. Logicalty, one is apt to suspect differential toxicokinetics. Analyses of metabolites are at present underway in this laboratory, but no appreciable dissimilarities between the two strains seem to exist in the distribution or excretion of TCDD [32]. In any case, disclosure of normal function of the resistance genes will undoubtedly markedly improve our understanding of the critical targets of TCDD toxicity. ACKNOWLEDGEMENTS

I wish to thank Professor Jouko Tuomisto and Dr. Outi Muona for valuable comments and advice. I am also grateful to Ms. Merja Lepp~m~ki and Mr. Jere Linden for technical assistance. This study was financially supported by the Academy of Finland, Research Council for Environmental Sciences (Grant No. 24/2X$ by the Finnish Cultural Foundation, Fund of Northern Savo, and by the Emil Aaltonen Foundation. REFERENCES I Poiand, A. and Knutson, J.C. (1982) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517-554. 2 Poland, A, and Glover, E. (3975) Genetic expression of aryi hydrocarbon hydtoxylase by 2,3,7,&tetrachlorodibenzo-p-dioxin: evidence for a receptor mutation in genetically non-responsive mice. Mol. Pharmacol. 11,389-398. 3 Vecchi, A., Sironi, M., Canegrati, M.A., Recchia, M. and Garattini, S. (1983) Immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in strains of mice with different susceptibility to induction ofaryl hydrocarbon hydroxylase. Toxicol. Appl. Pharmacol. 68,434-441. 4 Silkworth, J.B., Antrim, L. and Sack, G. (1986) Ah receptor mediated suppression of the antibody response in mice is primarily dependent on the phenotype of lymphoid tissue. Taxicol. Appl. Pharmacol. 86,380.390. 5 Poland. A., Greenlee, W.F. and Kende, AS. (1979) Studies on the mechanism of action of the chlorinated dibenz~~-diox~ns and related compounds. Ann. N.Y. Acad. Sci. 320.214230. 6 Hassaun, EA.-M. (198s) Tcratogcnicity and in vitro fetal thymus toxicity of 2,3,7,8-tetr~chlorodibenzo-p-dioxin and its congeners: segregation with the Ah locus. Acta Pharm. Suer. 22, 1’75-176. 7 Jones, K.G. and Sweeney, G.D. (1980) Dependence of porphyrogenic effect of 2,3,7,&tetrachlorodiben~o~)dioxin upon inheritance of aryl hydrocarbon hydroxykase responsiveness. Toxicol. Appl, Pharmacol. 53,4249. 8 Luster, M.I., Hong, L.H., Boorman, G.A., Clark G., Hayes, H.T., Greenlee, W.F., Dald, K. and Tucker, A.N. (1985) Acute myeiotoxic responses in mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Appl. Pharmacol. 81, 156-165. 9 Mohammadpour, H., Murray, W.J. and Stohs, S.J. (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced lipid peroxidation in genetically responsive and non-responsive mice. Arch. Environ. Contam. Toxicoi. 17,645-650. tO Nebert, D.W. (1986) The 1986 Bernard B. Brodie award lecture: The genetic regulation of drugmetabojizin~ enzymes. Drug Metab. Disposit. 16, f-8.

55

I I Kahl. G.F.,

Friederici,

sion of regulatory

D.E., Bigelow, SW.,

and structural

Okey, A.B. and Nebert,

gene products

associated

D.W. (1980) Qntugenetic

expres-

with the Ah locus. Dev. Pbarmacol.

Ther.

I, 137-162. I2 Goldstein,

J.A.

and

Hardwick,

J. (1984) Regulation

2,3,7,8-tetrachlorodibenzo-p-dioxin Biological Spring

Mechanisms

Harbor,

13 Durrin,

of a multigene

and related compounds.

of Dioxin Action.

Banbury

family

In: A. Poland

Report

of P-450

isozymes

and R. Kimbrough

18, Cold Spring

Harbor

by

(Eds.),

Laboratory,

Cold

NY, pp. 1 I%133.

L.K., Jones,

rodi~nzo-~-dioxin

P.B.C., Fisher, J.M., Galeazzi, mceptors

regulate

D.R. and Whitlock,

transcription

of the cytochrome

J,P. (1987) 2,3,7,&TetrachIoPi-450 gene. J. Cell. B&hem.

35, $53.-160. 14 Nebert,

D.W., Goujon,

cyclic hydrocarbons: I5 Gielen, J.E., Goujon, ase induction. 16 Thomas,

F.M. and Gielen, J E. (1972) Aryl hydrocarbon

simple autosomal F.M. and Nebert,

II. Simple Mendelian

P.E., Kouri,

duction

dominant

D.W. (1972) Genetic regulation

expression

R.E. and Hutton,

D.W.,

multigene

Negishi,

M., Lang,

family necessary

mnne system.

between

M,A.,

for survival

In: E.W. Caspari

@i.),

induction

by poly-

New Biol. 236, 107-I 10.

of aryl hydrocarbon

hydroxyl-

in mouse tissues in vivo. J. Biol. Chem. 247, 1125-I 137.

J.J. (1972) The genetics

in mice: a single gene difference

17 Nebert,

hyroxylase

trait in the mouse, Nature

C57BL/6J

Hjelmeland.

Biochem.

hydroxylase &net.

in-

6, 157-168.

L.M. and Eisen, H.J. (1982) The Ah locus, a

in a chemically Advances

of aryl hydrocarbon

and DBA/2J.

adverse

in Genetics,

environment:

comparison

Vol. 21. Academic

with the im-

Press, New York.

pp.

1-52. I8 Gasiewicz,

T.A. and Rucci, G. (1984) Cytosolic

dence for a homologous 19 Neal. R.A., Olson.

nature among

J.R., Gasiewicz,

chlorodibenzo-p-dioxin 20 Chapman,

D.E. and Schiller,

21 Knutson, 22 Greim,

for 2,3,7,~-tetra~h~orodi~nzo-~d~oxin. species. Mot. Pharmacol.

L.E. (1982) The toxieokinetics

Drug Metab.

C.M. (1985) Dose-related A. (1982) Response

Evi-

26,90-98. of 2,3,7&tetra-

Rev. 13, 355-385.

effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Appl. Pharmacol. of murine

78, 1477157,

epidermis

to 2,3,7,8-tetrachlorodibenzo-p-

of the Ah and hr loci. Cell 30. 225-234.

H. and Rozman.

(TCDD).

systems.

and DBAjZJ mice. Toxicol.

J.C. and Poland,

dioxin: interaction

receptor mammahan

T.A. and Geiger,

in mammalian

in C75BL/6J

(TCDD)

various

K. (1987) Mechanism(s)

of toxicity

of 2,3,7,8-tetrachlorodibenzo-p-dioxin

VDi Ber. 634,399427.

23 Pohjanvirta, receptor

R., Juvcnen,

R., Kareniampi,

S., Raunio,

S., H. and Tuomisto,

levels and the eRect of 2~3,7,~-tetra~hlorodi~nzo-~-dioxin

monooxygenase

activities

in a TCDD-susceptible

and -resistant

J. (1988) Hepatic

(TCDD) rat strain.

on bepatic

Toxic&.

Ah-

microsomai

Appf. Pharmaed,

92, 131.-140. 24 Pohjanvirta, ly resistant

R., Tuomisto, to TCDD.

J., Vartiainen,

I. Pharmacoi.

25 Siegel, S. (1956) Nonparametric

T. and Rozman,

Toxicol. Statistics

K. (1987) HaniWistar

rats are exceptional-

60, 145-150. far the Behavioral

Sciences.

McGraw-Hill

Kogakusha,

Tokyo. 26 Pohjanvirta,

R. and Tuomisto,

Arch. Toxicol.. 27 Sehwetz,

Suppl.

B.A., Norris,

C.G. (1973) Toxicology 28 Beatty, oxidase

(TCDD)

rats are exceptionally

J.M., Sparschu, of chiorinated

G.L.,

Rowe, V.K., Gehring,

dibenzo-p-dioxins.

Environ.

resistant

P.J., Emerson, Heaith

P.W., Vaughn,

W.K. and Neal, R.A. (1978) Effect of alteration

(MFO) activity

on the toxicity of 2,3,7,~-te~a~hiorodi~nzo-~-toxin

Pharmacol. 29 Walden.

J. (1987) Han/Wistar

to TCDD.

II.

11,344347. Perspect,

J.L. and Gerbig, 5,87-99.

of rat hepatic (TCDD).

mixed-function Toxicof.

45,513-519. R. and Schitler, in four (sub)-strains

C.M.

(1985) Comparative

toxicity

of adult male rats. Toxicol.

of 2~3,7,~-t~trachlorodi~nzo-~-dioxin

Appf. Pharmacol.

77,490-495.

Appf.

56

30 Pohjanvirta, R., Kulju, T., Morsek, A.F.W., Tuominen, R., Juvonen, R., Rozman, K., Mlinnisto, P., Coflan, Y., Sainio, E.-L. and Tuomisto, J. (1989) Target tissue morphology and serum bi~hemistry following TCDD exposure in a TCDD-susceptible and a TCDD-resistant rat strain. Fundam. Appl. Toxicol. 12 (in press). 31 Pohjanvirta, R., Tuomisto, L. and Tuomisto, J. (1988) TCDD is more toxic centrally than peripherally. In: Abstracts, 29th Congress of the European Society of Toxicology, Munich, September 4-7, 1988, p. 140. 32 Tuomisto, J. and Pohjanvirta, R,, (1988) Distribution and excretion of TCDD in Han/Wistar and Long-Evans rats. In: Abstracts, 29th Congress of the European Society of Toxicology, Munich, September 4-7, 1988, p. 42.