Reduced serum thyroid hormone levels in hexachlorobenzene-induced porphyria

Toxicology

Letters,

71

30 (1986) 71-78

Elsevier

TOXLett.

1528

REDUCED SERUM THYROID HORMONE LEVELS IN HEXACHLOROBENZENE-INDUCED PORPHYRIA (Hexachlorobenzene; induction) K. ROZMANab*, ‘Department Kansas

J.R.

toxicity; hypothyroidism;

GORSKIa,

of Pharmacology,

City,

(U.S.A.)

Umweltforschung

Miinchen,

October

14th, 1985)

received

November 2nd,

26th,

microsomal enzyme

and A. PARKINSON=

and Therapeutics, fiir

8042.Neuherberg

(Revision

December

Toxicology

66103 and bAbteilung

(Received (Accepted

P. ROZMANa

porphyria;

University

Toxikologie

of Kansas Medical

der Gesellschaft

fir

Center,

Strahlen-

und

(F. R. G.)

1985)

1985)

SUMMARY The effect of feeding 0.1% hexachlorobenzene porphyrin

excretion,

serum

in female Sprague-Dawley 33% mortality by dietary

thyroid

rats. This dosage

with a mean time to death

HCB,

whereas

(HCB) for 55 days on mortality,

hormones

non-survivors

and induction regimen,

followed

of 67 k 4 days.

underwent

a rapid

(4-fold)

and a significant

(Ts). When rats were returned prox.

100 times higher

the experiment the

activity

weight loss (wasting)

and

UDP-glucuronosyl

HCB-induced

lethality induction

benzo[a]pyrene

*To whom

c reductase

as substrate)

HCB,

hormones

cytochrome

were significantly hydroxylase

occur by different by HCB,

prior to death.

in the excretion continued

suppressed.

indicator

P-450 and cytochrome

induced,

03.50

whereas

the activity

(2) reduced

T4 and Tj serum levels

of aryl hydrocarbon

hydroxylase

(with

of HCB exposure.

T3, triiodothyronine;

Td, thyroxine;

Science Publishers

B.V. (Biomedical

Division)

of

that (I)

be addressed.

hexachlorobenzene;

0 Elsevier

bs and

aminopyrine-N-

chlorodibenzo-p-dioxin.

0378-4274/86/S

to rise (ap-

At the end of

was not. Results demonstrate

mechanisms,

and (3) induction

At the

of urinary

(Td) and triiodothyronine

porphyrins

remained

in

was not affected

pentoxyresorufin-0-dealkylase,

is not a sensitive

should

of urinary

of liver microsomal

transferase

and porphyria

correspondence

Abbreviations:

and serum thyroid

and benzo[a]pyrene

of porphyria

an increase

urinary

was studied

diet, resulted

of survivors

in the levels of serum thyroxine

diet the excretion

ethoxyresorufin-0-deethylase,

NADPH-cytochrome accompany

than controls)

(day 97), the concentration of

demethylase

decrease

to a regular

enzymes

by 42 days of a regular

Body weight

end of the dosing period (day 55), rats fed the HCB diet exhibited porphyrins

body weight,

of liver microsomal

TCDD,

2,3,7,8-tetra-

72 INTRODUCTION

Chronic exposure of laboratory animals and to HCB elicits a number of effects, such as induction of liver microsomal enzymes, porphyria, hypothyroidism, a wasting syndrome and lethality [l-3]. These manifestations of HCB exposure are quite similar to those seen in laboratory animals intoxicated with polychlorinated biphenyls or TCDD [4-61. The causal relationships between these responses to polyhalogenated aromatic hydrocarbons, as well as the ultimate cause of death, remain largely unknown despite many years of intensive investigation. It has been shown that the toxicity of halogenated aromatic hydrocarbons in rats and mice correlates well with their ability to induce liver microsomal aryl hydrocarbon hydroxylase (benzo~a]pyr~ne) and UDP-glucuronosyl transferase (ff-naphthol) activity [7]. However, the induction of these enzymes is apparently not responsible for the lethal effect of halogenated aromatic hydrocarbons [8]. To gain more insight into the mechanism of toxicity of HCB, this experiment examined the time course of various end points of toxicity (mortality, body weight, porphyria), thyroid hormone status and microsomal enzyme induction in rats chronically exposed to dietary HCB. METHODS

15 Female Sprague-Dawley rats (Sasco, Omaha, NE) were housed in metabolism cages with free access to feed (Ralston Purina Co., St. Louis, MO) and water (room temperature: 2.5 + 2°C; 12 h light/dark cycle and uncontrolled humidity). After 1 week, rats were divided into 2 groups. 6 rats (271 f 2 g), designated as controls, were maintained throughout this experiment under the conditions described above. For 55 consecutive days, 9 rats (272 + 5 g), were fed a diet containing 0.1 crloHCB (Teklad No. 83147, Madison, WI). HCB was purchased from Aldrich, Milwaukee, WI, and recrystallized from benzene until greater than 99% pure as determined by gas chromatography (Varian No. 3700, electron capture detector, SE-30 column, 30 ml/min nitrogen flow at 200°C). After the 55-day dosing period, the rats were returned to a regular diet for an additional 42 days. Body weight of each rat was measured daily and mortality recorded. 24-h urine samples were collected from 6 rats in each group at 41, 55, 62 and 97 days after the first dose of HCB and total porphyrins were determined. Urine (1 ml) was subjected to anion exchange chromatography (AGI-X8, 50-80 mesh, chloride form, Bio-Rad Laboratories, Richmond, CA) and the absorbance of the 3 N hydrochloric acid eluate was measured at 430, 380 and 403 nm. The absorbance was corrected for the presence of biological material and recovery validated as described by Harmsen and Strik [9]. Blood (1 ml) was drawn from the tail vein of 6 rats in each group at 55, 62 and 97 days after the first dose of HCB, and T4 and T3 levels in serum were determined by radioimmunoassay (Corning Reagents Nos. 474207 and 474209).

73

Survivors of the experiment were killed 97 days after the first dose of HCB and livers were removed and weighed. Liver microsomes were prepared as described by Lu and Levin [lo] and stored as a suspension in 0.25 M sucrose at - 80°C. Protein concentration was measured by the method of Lowry et al. [ll] with bovine serum albumin as standard. Cytochrome P-450 content was determined according to Omura and Sato [ 121; from the carbon monoxide-difference spectrum of dithionite-reduced microsomes based on an extinction coefficient of 91 mM - ‘cm - ‘. The concentration of cytochrome bs was determined by the method of Raw and Mahler [13]; from the difference spectrum between NADH-reduced and oxidized microsomes based on a corrected extinction coefficient of 185 mM - ‘cm - ’ [ 121. The rate of oxidative N-demethylation of aminopyrine was measured essentially as described by Parkinson and Safe with minor modifications [14]; from the production of formaldehyde by measuring absorbance at 412 nm after reaction with Nash reagent [15]. Activities of benzo[a]pyrene hydroxylase [16] and 7-ethyoxyresorufin 0-deethylase [17] were measured as described by Corski et al. [18]. The 0-dealkylation of 7-pentoxyresorufin (Pierce Chemical Company) was measured essentially as described [19]. The 7-pentoxyresorufin was added to the l-ml incubation mixture in 40 ~1methanol at a final concentration of 5 FM. NADPH-cytochrome c reductase activity was measured as described by Phillips and Langdon [20] with lo-20 pg/ml of microsomal protein. UDP-glucuronosyl transferase activity towards I-naphthol was measured according to Otani et al. [21]. Microsomes were solubilized with 10 mM 3[(3-cholamidopropyl)-dimethylammonio]l-propane sulfonate [22] and the reaction mixtures (0.5 ml) were incubated at 37°C in the presence of Tris-HCl (0.2 M, pH 7.4), saccharic acid-1,blactone (2.2 mM), MgC12 (10 mM) and [i4C]naphthol (0.5 mM) (New England Nuclear Corp., Boston, MA) at the final concentrations indicated. Reactions were started by the addition of 2 pmol UDPglucuronic acid. Reagent blanks contained no UDP-glucuronic acid. Reactions were terminated by the addition of 0.5 ml of ice-cold ethanol. In preliminary experiments, conditions were established such that the amount of product formed was proportional to both incubation time and microsomal protein concentration. Statistics were performed by Student’s two-tailed t-test [23]. RESULTS

Of 9 rats fed a diet containing 0.1% HCB for 55 days, 3 died approx. 2 weeks after being returned to a regular diet. As shown in Fig. 1, body weight of survivors and non-survivors was not different from controls at the time HCB administration was discontinued (day 55). However, non-survivors started losing weight at about day 60 in a fashion resembling TCDD-induced ‘wasting syndrome’ [24]. 41 Days after the first dose of HCB, excretion of total urinary porphyrins was not different in control and HCB-treated rats. At the time rats on the HCB diet were

10

20 DAY

30

Fig. 1. Body weight changes hexachlorobenzene treated)

40

AFTER

(HCB)

50

FIRST

DOSE

60

70

OF HCB

in female Sprague-Dawley for 55 days.

Values

represent

rats fed a regular the mean

f

diet or a diet containing

S.E. of 6 (controls)

0.1%

or 9 (HCB-

rats.

returned to regular rat chow (day 55), excretion of urinary porphyrins was mildly elevated. In spite of cessation of dosing, excretion of urinary porphyrins in survivors of the HCB diet continued to increase and, after 4 weeks, was almost 100 times greater in treated rats than in controls (Table I). At the time of onset of increased porphyrin excretion in urine of HCB-treated rats (day 55), both serum Tq and T3 were significantly reduced (Table II). Serum thyroid hormones remained suppressed during 42 days of subsequent monitoring (Table II). A minor rebound effect appeared to take place between days 62 and 97 in terms of serum T4. At termination (day 97) both total cytochrome P-450 and cytochrome bs content were elevated in liver microsomes from HCB-treated rats compared to controls (Table III). The activity of ethoxyresorufin 0-deethylase (lo-fold), pentoxyresorufin 0-dealkylase (12-fold), aminopyrine N-demethylase (less than 2-fold) and UDP-glucuronosyl transferase (2-fold) were significantly induced in rats fed the HCB-containing diet. In contrast, the activity of liver microsomal NADPHTABLE

I

EXCRETION

OF TOTAL

FED A DIET CONTAINING Day after first dose of HCB

URINARY 0.1%

PORPHYRINS

Total

urinary

Controls

a Mean

f

porphyrins

SPRAGUE-DAWLEY

(HCB)

FOR 55 DAYS

&g/day) HCB

41

3.2 + 0.1”

4.4 f

55 62

3.0 f 0.3 2.9 + 0.5

11.6 + 37.9 +

91

1.5 rt 0.3

SE; n=6.

* Significantly

IN FEMALE

HEXACHLOROBENZENE

different

from controls

(P
120

0.9 1.7* 8.7*

* 21*

RATS

75

TABLE

11

SERUM

LEVELS

OF

SPRAGUE-DAWLEY

THYROXINE

(Td)

AND

TRIIODOTHYRONINE

RATS FED A DIET CONTAINING

0.1%

(Ts)

IN

FEMALE

HEXACHLOROBENZENE

(HCB)

FOR 55 DAYS Day after

first

T, (ng/dl)

T4 (Irg/dl)

dose of HCB Controls

HCB

Controls

HCB

55

3.8 + 0.2a

< 1.0

64.2 * 2.0

48.1 f

62

4.3 + 0.3


74.8 * 2.8

51.3 + 1.5*

4.1 * 0.3

1.5 f 0.3*

75.0 + 5.3

53.0 f 4.1*

97 a Mean

3.2%

& SE; n=6.

*Significantly

different

from controls

(P
cytochrome c reductase and benzo[a]pyrene hydroxylase was not altered by dietary HCB at termination of the experiment (day 97). DISCUSSION

This experiment confirms an earlier report of Ockner and Schmid [25], that, in TABLE LIVER

111 MICROSOMAL

ENZYME

DIET CONTAINING Microsomal

0.1%

INDUCTION

IN FEMALE

HEXACHLOROBENZENE

enzyme

SPRAGUE-DAWLEY

Enzyme

content/activitya

Controls Cytochrome (nmol/mg

P-450

(nmol/mg

1.01 +

0.09*

bs

0.44 + 0.04

0.69 *

0.02*

0.33 + 0.01

0.32 f 0.07

0.42 t 0.04

0.46 +

protein)

NADPH-cytochrome &mol/mg

c reductase

protein/min)

Benzo[a]pyrene (nmol/mg

hydroxylase

0.05

protein/min)

Ethoxyresorufin (pmol/mg

0deethylase

27.1

f 2.2

260

+ 57*

20

13

240

f 40*

protein/min)

Pentoxyresorufin (pmol/mg

0-dealkylase

protein/min)

Aminopyrine

N-demethylase

6.5

& 0.7

85.6

+ 5.4

10.1

& 0.1*

protein/min)

UDP-glucuronosyl transferase (I-naphthol) (nmol/mg protein/min) a Determined b Mean

HCB

0.70 + 0.05b

protein)

Cytochrome

(nmol/mg

RATS FED A

(HCB) FOR 55 DAYS

97 days after

first dose of HCB.

+ SE; n=6.

* Significantly

different

from controls

(P< 0.05).

161

k 24*

76

rats, the lethal effects of chronic HCB feeding (0.2% of diet for 4 weeks) are poorly correlated with disturbances of porphyrin metabolism. In the present study, we observed that non-survivors with a relative minor disturbance of porphyrin metabolism started wasting away 5 days after discontinuing the HCB diet, whereas survivors did not show any loss of body weight during the 42-day post-exposure period, even though they developed severe porphyria (Fig. 1 and Table I). These findings indicate that the mechanisms of HCB-induced mortality and porphyria are different. Induction of porphyria by HCB occurred as described in more detail by Koss et al. (261. Serum thyroid hormone levels were significantly reduced at the time of onset of porphyria regardless of the survival status of the rats (Table II). Both serum T4 and T3 levels remained suppressed during the 42-day period after cessation of the HCB diet without an apparent adverse effect on rat body weight (Fig. 1). Therefore, reduced serum T4 hormone levels may not necessarily be viewed as a sign of toxicity. However, decreased serum T4 has been shown to occur after exposure of rats to several chlorinated aromatic hydrocarbons that are capable of inducing porphyria [1,5,6,27-291. Based on available data, it is not possible to determine whether reduced serum thyroid hormone levels and porphyria are causally related. The activities of ethoxyresorufin 0-deethylase and pentoxyresorufin 0-dealkylase were extremely sensitive indicators of the effects of dietary exposure to HCB on rat liver microsomal cytochrome P-450. The induction of cytochrome P-450~ by HCB was apparent from a lo-fold induction of ethoxyresorufin 0-deethylase activity, although the activity of benzo[a]pyrene hydroxylase was unaffected by HCB treatment. The difference in the apparent inducibility of these enzyme activities presumably reflects the ability of cytochrome P-450 isozymes other than cytochrome P-450~ to hydroxylate benzo[a]pyrene, but not ethoxyresorufin [30]. The induction of cytochrome P-450b by HCB was apparent from a 1Zfold induction of pentoxyresorufin 0-dealkylase activity, as well as a modest increase in aminopyrine N-demethylase activity. It was shown recently that the dealkylation of pentoxyresorufin is preferentially catalyzed by cytochrome P-450b and is highly inducible by phenobarbital treatment [ 191. These data (Table III) corroborate results of recent immunochemical studies [3 l] indicating that HCB is both a phenobarbitaltype inducer of rat liver microsomal cytochrome P-450b and a 3-methylcholanthrene-type inducer of cytochrome P-450~. In conclusion, these data in conjunction with other experiments [8] suggest that correlations between microsomal enzyme induction and toxicity may be misleading and should not be used without understanding of the underlying mechanism of toxicity.

77

ACKNOWLEDGEMENT

A.P. gratefully acknowledges the financial support Flossie West Foundation and NIH grant ES 03765.

of the PMA, Speas and

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