Relationship between survival times of rats exposed to lethal level of nitrogen dioxide and arylhydrocarbon hydroxylase activity in lungs

Toxicology Letters,

233

19 (1983) 233-239

Elsevier

RELATIONSHIP RETWEEN SURVIVAL TIMES OF RATS EXPOSED TO LETHAL LEVEL OF NITROGEN DIOXIDE AND ARYLHYDROCA~ON HYDROXYLASE ACTIVITY IN LUNGS (NOz; survival times; arylhydrocarbon

hydroxylase;

lungs)

MASARU SAGAI, SEIICHI SUZUKI and TAKAMICHI ICHINOSE Basic Medical Sciences Division,

The National Institute for Environmental

Studies, P.O.

Tsukuba,

Ibaraki 305 (Japan)

(Received May 3Oth, 1983) (Accepted July 19th, 1983)

SUMMARY A previous paper has shown that survival times of rats exposed to lethal levels of NO2 were prolonged by administration of enzyme-inducing agents prior to NO2 exposure, and that the survival time of female rats was longer than that of male rats. In order to investigate a mechanism on prolongation of survival times by administration of enzymeinducing agents and on sex-difference, a relationship between survival times and microsomal enzyme activity in lungs of rats administered enzyme-inducing agents was examined. The survival times of rats exposed to 65 ppm NO2 were prolonged with increase of 3MC doses, and AHH activity in iungs of rats rose with increase of 3MC doses. A significant correlation between the survival time and AHH activity was observed. Induction of AHH activity of female rats administered 3MC was higher than that of male rats. NADPH-dependent cytochrome c reductase activity in lungs of male and female rats did not change with 3MC administration, and a significant relationship between the survival time and NADPHdependent cytochrome c reductase activity was not observed. These results suggest that cytochrome Pi-450 system in lung microsomes may have protective action against the toxicity of Not.

INTRODUCTION

With regard to the induction of drug-metabolizing enzymes in liver and kidney, a considerable amount of data have been reported. Recently, many data on the induction or reduction of drug-metabolizing enzymes in lungs are reported by Law et

Abbreviations:

AHH, arylhydrocarbon

hydroxylase; 3MC, 3.methylcholanthrene.

037&4274/83/S 03.00 @ Elsevier Science Publishers R.V.

234

al. [l] using guinea

pigs, Burke and Prough

[2] using hamsters

and Seifreid

[3] using

mice. The assay of benz(a)pyrene convenient

tool to follow

hydroxylase the induction

(AHH)

activity

of the cytochrome

has been used as the most Pi-450

mixed

function

oxidase system. It is well known that the induction of drug-metabolizing enzymes by 3MC shows different response among some tissues [4, 51, and sex-related differences [5, 61. Recently, Warren and Bellward [7] reported the complete doseresponse curves of AHH in liver, kidney and lung tissues of both male and female rats. They observed that lung tissue was the most sensitive to the induction, followed by liver and kidney in all cases. It is reported that AHH activity in lungs is markedly affected by air pollutants such as NOz, ozone or SO2 [8, 91. On the other hand, we reported previously that the survival times of rats exposed to lethal levels of NO2 were prolonged by administration of some enzyme-inducing agents prior to the exposure to NOz, and that the survival time of male rats to NO2 was longer than that of female rats [lo]. In order to investigate a mechanism on prolongation of survival times by administration of 3MC and on sex-related differences in the present study, a relationship between the survival time and AHH activity in lungs of rats administered 3MC was examined.

MATERIALS

AND

METHODS

Animals, 3MC, administration and NO2 exposure. JCL: Wistar rats, 8 weeks old, were used. To determine the mortality, each group of 20 rats was exposed to 30, 35, 40, 45, 50, 55 and 60 ppm NO2 for maximum 8 h, respectively. Mortality ratios were counted from the numbers of rats that died within 3 days after the start of NO2 exposure. To examine the effect of 3MC on survival times (min), i.e., the time from the start of exposure to 65 ppm NO2 until the death of the rats, male and female rats were administered 3MC of 2.5, 5, 10, 20 and 40 mg/kg body wt. i.p. for 4 consecutive days, and NO2 exposure was conducted on day 5. 3MC was dissolved in olive oil, and the administration volume of olive oil containing equivalent doses of 3MC was 2 ml/kg body wt. in all groups. The control rats were given only olive oil. NO2 exposure was conducted according to the previous report [lo]. For assay of AHH activity in lungs, the rats administered 3MC of each dose of 4 consecutive days were killed on day 5 under light ethyl ether anesthesia by removing blood from the neck artery, and then the lungs were perfused via the right ventricle with isotonic saline. Enzyme preparation. Lungs were homogenized in a glass-Teflon homogenizer under Nz, and a 10% homogenate was made with 50 mM Na, K-phosphate buffer (pH 7.5), which was deaerated under reduced pressure and bubbled with N2. The homogenate was centrifuged at 200 x g for 5 min, and then the upper layer was

235

recentrifuged at 10000 x g for 20 min. The supernatant was again centrifuged at 105 000 x g for 60 min, and the obtained pellets were suspended in 30%-glycerol125 mM Tris-HCl buffer (pH 7.4) by gentle homogenization to give a final protein concentration of 8-12 mg/ml. Enzyme assay. The requirements for linearity and optimal benz(a)pyrene hydroxylase (AHH) activity in the lungs were determined by a modification of the method of Warren and Bellward [7]. The final volume of the incubation mixture was 1 ml and contained 1.08 pmoles each of NADH and NADPH, 9 pmoles MgCL, 1.8 mg bovine serum albumin, 450 ~1 enzyme preparation, 80 nmoles benz(a)pyrene in 50 ~1acetone and 25 pmoles Tris-HCl buffer (pH 7.4)-30% glycerol. The incubation was performed at 37°C for 15 min. The extraction of 3-hydroxy benzpyrene was done by the method of Nebert and Gelboin [4], and the amounts of 3-hydroxy benzpyrene was determined with a recording spectrophotofluorometer (Hitachi-560) with a slit width of 10 nm and with an excitation wavelength of 396 nm and emission wavelength of 522 nm. NADPH-dependent cytochrome c reductase activity was determined by the method of Strobe1 et al. [ll]. Proteins were measured by the method of Lowry et al. [12] using bovine serum albumin as a standard.

RESULTS

We reported previously that there is a sex difference on susceptibility as expressed by survival times of Sprague-Dawley rats exposed to lethal levels of NO2 [lo]. In the present study, to investigate whether other strains of rats also show similar

300c

o-/'30 35 j !I 40 45 '

50 55 60

NO2concentration ( PM~ 1

14/ 8

0 I 3 Fig. 1. Mortality Female

curves

of rats exposed

to lethal levels of nitrogen

2.5 5 MC Dose

dioxide

(

10

20

ms/ks B,W.

40

)

for 8 h. l , Male rats;

0,

rats.

Fig. 2. Relationship between Female rats. M + S.E.

3MC doses and survival

times by 65 ppm NO> exposure.

l , Male rats; 0,

236

results

on sex difference,

Wistar

male and female

rats were exposed

45, 50, 55 and 60 ppm NO2 for 8 h, and the mortality the numbers

of rats that died within

to 30, 35, 40,

ratios were determined

3 days after the start of NO2 exposure

from (Fig. 1).

lOO%-lethal concentration (LCIoo) of male rats was 50 ppm NO2, and that of female rats was 55 ppm NOz. 50%-Lethal concentration (LCSO) were extrapolated from Fig. 1 to be 39 ppm for male rats and 45 ppm for female rats. This result also shows that the sensitivity of male rats to NO2 was higher than that of female rats. The survival times (min) of rats administered 3MC of 0, 2.5, 5, 10, 20 and 40 mg/kg body wt. prior to the exposure to 65 ppm NO2 were determined (Fig. 2). The survival times of male rats increased linearly until 10 mg of 3MC/kg body wt., but those of male rats given 3MC of 20 or 40 mg/kg body wt. were decreased. On the other hand, the survival times of female rats were prolonged linearly with increases of 3MC doses. This result shows that a mode of dose-response relationship is different between male and female rats. To investigate a mode of enzyme induction by 3MC, benz(a)pyrene hydroxylase (AHH) activity was examined. Dose-response relationship between 3MC dose and AHH activity in lung microsomes is shown in Fig. 3. The dose-response curve of AHH activity in lungs of male rats remained at a lower level than that of female rats. AHH activity in the control group of male rats (0.25 U/mg protein/min) was lower than that of female rats (0.78 U/mg . protein/min). These dose-response curves showed a similar pattern to the survival times as shown in Fig. 2. The maximum induction of AHH activity in male rats was observed at 5-10 mg of 3MC/kg body wt., and the activity of male rats given 3MC of 20 mg or 40 mg/kg body wt. was lowered. On the other hand, the AHH activity of female rats was induced linearly with increase of 3MC dose without such limitation in male rats. From the results in Figs. 2 and 3, the correlation between the survival times against NO2 exposure and the AHH activity in lungs of rats administered 3MC prior to the exposure to 65 ppm NO2 was examined and depicted in Fig. 4. A significant correlation between the survival times and AHH activity was observed with high correlation

coefficients

(r = 0.9288,

PC 0.001).

50 40

30

h

20

'!/ lOhc""fi o

I

7.5

5

3 MC Dose

Fig. 3. Relationship

10 (mg/kg

between

20 B.W.

40

)

3MC and AHH

activity

in lungs. l , Male rats; 0; , Female rats. M + S.E.

237

"

120

140

160

180

200

220

Survival Time (min

240

260

280

)

Fig. 4. Relationship between AHH activity in lungs and survival times by 65 ppm NO2 exposure. l , Male rats; 0, Female rats. M 1 S.E. Correlation coefficient (r) between survival times and AHH was 0.9288 (P
Ok 0

2.5

5

10

20

3MC Dose (mg/kg B,N

40

f

Fig. 5. Relationship between 3MC doses and cytochrome c reductase activity in lungs. 0, Male rats; Female rats.

0,

The activity of NADPH dependent cytochrome c reductase which provide electrons to cytochrome PI-450 in microsomes was examined by using male and female rats given various doses of 3MC (Fig. 5). The activity of NADPH dependent cytochrome c reductase in lungs did not show any significant changes from the control level by 3MC administration. Furthermore, a significant correlation between the activity and the survival times was not observed. DISCUSSION

In the present study, it was observed that the survival times of rats exposed to

238

lethal level of NO2 were prolonged with increases of 3MC doses and that the prolonged survival times were correlated closely with the induction of AHH activity in lung microsomes. Furthermore, the evidence that the survival was longer than that of male rats and AHH activity of female

time of female rats rats was also higher

than that of male rats was shown. The sex-differences of AHH activity in lung microsomes were examined by Nebert and Gelboin [4] using six strains of mice, and they found that the enzyme activity of female mice was several times higher than that of male mice. On the other hand, Gurtoo and Parker [S], and Chharbra and Fouts [6] reported that there is no difference in the activity between male and female rats. However, recently, Warren and Bellward [7] reported that AHH activity of female rats was 3 times higher than that of male rats, and that the induction of AHH activity by 3MC administration was marked in female rats rather than in male rats. Furthermore, they found that AHH activity in liver and kidney was not induced by 3MC of 5 mg or less/kg body wt., and that the dose response relationship in liver and kidney were markedly different from the dose response relationship in lungs. Our present result also showed that AHH activity in lungs of rats administered 3MC of 2.5-5 mg/kg body wt. was induced significantly higher than that of the control rats. Furthermore, our present results showed that the survival times of rats exposed to the lethal level of NO2 correlate with AHH activity in lungs rather than that in iiver and kidney. With regard to the changes of AHH activity by NO2 exposure, Palmer et al., [13] reported that AHH activity in the tracheobronchia mucosa of rabbits exposed to 5, 20 and 40 ppm NO1 for 3 h did not change. On the other hand, Husain and Dinhan [9] reported that AHH activity in lungs of rats exposed continuously to 14.7 ppm NO2 for 4 weeks increased 1.5-4 fold. These results show that AHH activity was not altered by short term exposure even to high concentrations of NOz, but that it was induced markedly by long-term exposure of NOz. We reported previously that the antioxidative protective systems including glutathione peroxidase in lungs of rats exposed to 0.4, 1.2 and 4 ppm NO2 was induced to maximum level at the 4th week, and that the formation of cytotoxic lipid peroxides was returned to minimum level [14]. These results suggest that cytochrome PI-450 as the true form of AHH enzyme may have a protective function against the toxicity of NOz. The investigation of the correlation between enzyme-induction by various agents and survival times might cast further

light on a protective

mechanism

against

toxicity

of NOz.

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