Mutation Research, 262 (1991) 125-128 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0165-7992/91/$ 03.50 ADONIS 016579929100008M

125

MUTLET 0458

A genetic study of human interferon-a-induced repair of DNA damage in hepatitis B patients Peilin W a n g 1, S h u y i n g C o n g 2, X i a o y o n g D o n g 1, Z a i c h u n C h e n 3 a n d C h u i p i n g M a 2 Departments of 1Biology and 3infectious Diseases, Tsingtao Medical College and 2Hospital of Infectious Diseases, Tsingtao (People's Republic of China) (Received 7 September 1990) (Accepted 27 September 1990)

Keywords: Hepatitis B; Human interferon-or;Sister-chromatid exchange; Repair of DNA damage

Summary In vitro treatment with human interferon-a (HuIFN-a) of hepatitis B virus-infected peripheral lymphocytes from 17 hepatitis B patients induced a decrease in the frequency of sister-chromatid exchanges (SCE). There was a significant difference in mean SCE frequencies between the HuIFN-o~-treated patients and the control group, but not between acute and chronic hepatitis B patients treated with HuIFN-c~.

There are many reports in the literature about DNA damage in hepatitis B patients and in children born after their parents had contracted hepatitis B (Ghosh, 1982; Liu, 1982; Wang, 1986b,c). However, little is known about the repair of DNA damage in drug-treated hepatitis B patients. The antiviral and antineoplastic effects of human interferon-a (HuIFN-c0 have been studied, and it has been used to treat hepatitis B (Hashida et al., 1985; Alexander et al., 1986; Borcky, 1986; Spiegel, 1987; Liu et al., 1988), but it is not known how HuIFN-c~ influences the repair of DNA damage. Correspondence: Dr. P. Wang, Department of Biology, Tsingtao Medical College, Tsingtao (People's Republic of China).

Materials and methods Materials

Seventeen hepatitis B (HB) patients (12 men and 5 women) with a mean age of 30.8 years entered our study. Seven of the individuals were defined as acute cases and 10 individuals were afflicted with chronic hepatitis. None of the patients had been exposed to any known mutagens or radiation for 6 months before analysis and no other diseases complicated the hepatitis infection, except that one individual had chronic bronchitis. The 6 serologic indexes of all patients were tested before analysis. Hepatitis B surface antigen (HBs-Ag) of all cases was positive; hepatitis Be antigen (HBeAg) was positive in 10 cases, anti-HBe in 5, anti-HBc in 14

126 032

and polyhuman serum albumin (PHSA) in 4. More than 3 serologic indices were positive in 14 cases.

0.28

~

Methods

0 m

o

Methods of cell culture, slide preparation, sisterchromatid differentiation and SCE counting have been described before (Wang, 1986a). Peripheral lymphocytes from HB patients were divided into 2 groups (HIA and HIB). HulFN-o~ (obtained from the Chengdu Institute of Biological Products under the Ministry of Public Health) was added to the culture media at different concentrations: 5000 IU/ml for group HIA and 50 IU/ml for group HIB. Peripheral lymphocyte cultures from the control group (HC) did not receive any HulFN-c~. Data were statistically evaluated using a paired ttest.

Results

The SCE frequencies observed in each experimental group and the distribution of mean SCE frequencies are shown in Table 1 and Fig. 1 respectively. Compared to previous data (Wang, 1986a), the mean SCE frequency of the HC group showed a significant difference ( P < 0 . 0 1 ) but the HB patient group did not ( P > 0.05). The whole experimental group (HIA and HIB) had a mean SCE frequency of 7.1255_+ 1.4464 per metaphase and

E

0.24 0.20

0 t_

5

o.~6

0000 O~ 0

t_

~

&... O--

o12

w

u

008 0.04 HIC

I HIA

k HIB

Fig. 1. Distribution of mean SCE frequencies in lymphocytes of HB patient groups.

0.1552_+0.0316 per chromosome. There was a highly significant difference between the experimental group and the control group ( P < 0.01). The SCE frequencies observed in acute and chronic HB patients are shown in Table 2. There were no significant differences in mean SCE frequencies between acute (control and treated) and chronic (control and treated) patients.

Discussion

SCE is a test method which can detect human DNA damage and repair of DNA damaged by hepatitis B virus (HBV) infection. The SCE fre-

TABLE 1 MEAN FREQUENCIES OF SCE IN LYMPHOCYTES OF HB PATIENT GROUPS Group

Cases

SCEs (S +_SD)/chromosome

SCEs (~ ± SD)/metaphase Cells

SCEs

SCE rate

P

Control

16

320

3481

10.8781 ± 2.7776

A

17

332

2402

7.2275 ± 1.8977

0.01

7.0100 + 1.9462

0.005

B

15

300

2103

Chromosomes

SCEs

SCE rate

14692

3481

0.2369 ± 0.0605

15264

2402

0.1575 +0.0414

0.01

0.1527 _+0.0424

0.005

13793

2103

P

127

TABLE 2 M E A N F R E Q U E N C I E S OF SCE IN L Y M P H O C Y T E S OF A C U T E AND C H R O N I C HB P A T I E N T S Gr oup

Cases

SCEs (x_+ S D )/ c hromos ome

SCEs (~ ± SD)/metaphase Cells

SCEs

SCE rate

P

Chromosomes

SCEs

SCE rate

-

5514

1270

0.2203 ± 0.0958

P

Acute Control

6

120

1270

10.5833 ± 4.4035

A

7

132

892

6.7767 ± 2.6570

0.005

6080

892

0.1471 ±0.0578

0.005

7.0000 ± 2.9175

0.01

5516

840

0.1482 ± 0.0637

0.01

11.0550 ± 3.5540

-

9178

2211

0.2408 ± 0.0774

7.5500 ± 2.4785

0.01

9194

1510

0.1647 ± 0.0541

0.01

7.0167 ± 2.5177

0.005

8277

1263

0.1528 ± 0.0548

0.005

B

Chronic Control

A

B

6

10

10

9

120

200

200

180

840

2211

1510

1263

quencies of peripheral lymphocytes in HB patients and children (including twins) born after their parents contracted HB have been reported (Wang, 1986b,c) to be significantly higher than those of normal children, adults and children born prior to their parents' contraction of the disease. IFN is a biologically active polypeptide produced by animal cells after stimulation with a variety of inducers; it is used for antiviral, antineoplastic and immunomodulating functions and to improve liver function in clinical therapy. Although IFN has been known since 1957 (Isaacs and Lindenmann, 1957), our molecular understanding of it is more recent. There are 3 genotypes (o~, 3 and 30 (Borcky, 1986). Studies of hybrid human-mouse cells have suggested that the ~ and 3 genes are located on the 9p (13-24) band of chromosome, and that of IFN-3, in man is located on chromosome 12 (Blumberg, 1986). At present, it is generally agreed that the antiHBV substances are badly needed. IFN offers a new approach to the therapy of HB which differs in

mechanism and spectrum of activity from the chemotherapeutic drugs presently used for hepatitis B patients. It is considered that the effectiveness of IFN therapy in the clinic will be greater when IFN is combined with synergistic drugs (Borcky, 1986). The present test showed that the mean SCE frequency in the HC group was significantly higher than in the HIA and HIB groups. This result demonstrated that the mean SCE frequency in peripheral lymphocytes of hepatitis B patients in vitro may be decreased by HuIFN-c~ and that H u I F N - a induced the repair of human DNA damaged by HBV. The result was consistent with that o f Suzuki (1986). The mean SCE frequency was higher in the HIA group than in HIB, but this difference was not significant (P>0.05). This suggested that the different concentrations of HuIFNused in this study do not have a significantly different effect on mean SCE frequencies. It further showed that increasing the dosage of HuIFN-ct might not have a beneficial effect on the repair of

128

DNA damage in HB patients. This corresponds with clinical research results (Weimar, 1988). Statistical comparison of the mean SCE frequencies of the groups indicated: (a) a highly significant difference (P0.05) between the acute and the chronic control, HIA and HIB groups. This suggests that there were no significant differences in mean SCE frequencies in peripheral lymphocytes of acute and chronic HB patients, that mean SCE frequencies were decreased and repair synthesis of human DNA damaged by HBV was induced, by treatment with HuIFN-c~. Our study shows that the mean SCE fequencies in peripheral lymphocytes of HB patients are reduced and that the genetic effect of repair of HBV-caused DNA damage is induced by HuIFNct. Future studies will show whether interferon, either alone or in combination with synergistic drugs, will be beneficial in the treatment of HBVinduced DNA damage. The mechanisms underlying DNA repair by interferon and various other agents (chemotherapeutic or herbal) will be investigated theoretically and genetic studies may provide experimental indices for the treatment of hepatitis B, the selection of suitable medication and the evaluation of therapeutic results in clinical practice.

References

chronic hepatitis B, Med. Foreign Country, Sect. Epidemiol. Contag. Dis., 14, 88-89. Blumberg, B.S. (1986) Model of primary hepatocellular carcinoma resulted in hepatitis B, Med. Foreign Country, Sect. Epidemiol. Contag. Dis., 13,210-212. Borcky, L. (1986) Current view on the perspectives of interferon therapy, Acta Virol., 30, 161-169. Diaz, M.D., et al. (1986) Interferon (IFN) and c-ets-1 gene in the translocation (9;11)(p22;q23) in human acute monocytic leukemia, Science, 231,265-267. Hashida, T., et al (1985) Therapeutic results of chronic and active hepatitis in child treated with human leukocyte interferon, Med. Foreign Country, Sect. Epidemiol. Contag. Dis., 2, 277-278. Heddle, J.A. (1982) Mutagenicity. New Horizons in Genetic Toxicology, Academic Press, New York, pp. 172-204. Jiang, Z.S., et al. (1981) DNA repair: a new realm of medical genetics, Med. Foreign Country, Genet. Sect., 4, 235-245. Liu, R., et al. (1988) An observation of lately therapeutic result of chronic and persistent hepatitis treated by human leukocyte interferon, Chin. J. Infect. Dis., 6, 54-55. Lu, C.Q. (1988) A study of association of primary hepatocellular carcinoma with hepatitis B, Chin. J. Epidemiol., 9, 5-7. Sorensen, P.J., et al. (1981) Effect of human leukocyte interferon on human lymphocytes in vitro: cytogenetic studies, Mutation Res., 90, 143-147. Spiegel, R.J. (1987) Clinical overview of alpha interferon studies and further directions, Cancer, 59, 626-631. Suzuki, N. (1986) Effects of human interferon-c~ on UV-induced DNA-repair synthesis and cell killing, Mutation Res., 175, 189-193. Thomas, H.C., et al (1986) Homology between HBV-DNA and a sequence regulating the interferon-induced antiviral system: possible mechanism of persistent infection, J. Mol. Virol., 19, 63-69. Vandenbussche, P. (1981) Enzymatic activities induced by interferon in human fibroblast cell lines differing in their sensitivity to the anticellular activity of interferon, Virology, 111, 11-22. Wang, P., et al. (1986a) Study of sister-chromatid exchange in peripheral lymphocytes of hepatitis B patients with HBsAG positive, Acta Acad. Qingdao, 22, 19-22. Wang, P., et al. (1986b) A study of sister-chromatid exchange in peripheral lymphocytes of hepatitis B patients with HBsAg positive and their children (1), Mutation Res., 175,249-254. Wang, P., et al. (1986c) A study of sister-chromatid exchange in peripheral lymphocytes of hepatitis B patients with HBsAg positive and their children (2), Acta Genet. Sin., 13, 144-149.

Alexander, G., et al. (1986) New view of antiviral treatment on

Communicated by J.M. Gentile

Acknowledgements We are indebted to Dr. Sandy Harrison and Professor W. Yongxun for their critical reading of the manuscript. We are grateful to Mr. L. Naiyuan for his technical assistance during the course of the experiments.

A genetic study of human interferon-alpha-induced repair of DNA damage in hepatitis B patients.

In vitro treatment with human interferon-alpha (HuIFN-alpha) of hepatitis B virus-infected peripheral lymphocytes from 17 hepatitis B patients induced...
246KB Sizes 0 Downloads 0 Views