Journal of Huazhong University of Science and Technology [Med Sci] 22 (1): 12-13, 2002

Effect of A-L Tonic Capsule on DNA Content in Rat Experimental Hepatocarcinogenesis GUAN Yang ( '~ Fa), ZHOU Zebin ( N ~ R . ) , ZHANG Chunming (~gr-~2]), RUAN Youbing (D.@;;~), WU Zhongbi (~(, ,5 ~ ) Department of Ultrastructural Pathology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030

Summary: The effects of A-L tonic capsule on DNA content in rat experimental hepatocarcinogenesis induced by diethylnitrosamine (DENA) were observed. The experimental rats were divided into 4 groups. With exception of group D in which the rats were only administered with DENA, the rats in the groups A, B, C were previously, simultaneously and subsequently fed with A-L tonic capsule re- spectively while they were administered with DENA. The DNA content of all rat livers was measured using automatic image analysis technique 20 weeks after administration of DENA. The results showed that the highest and lowest DNA contents were respectively seen in the groups D and A. There was significant difference in DNA contents between the groups A or B or C and D, and also between the groups A and B or C (both P<~0.01). 4 components (4C) and ~ 5 C cells were predomi- nant in the group D, while 2C cells were the minority. The number of 2C cells in the groups A, B, C was significantly higher than that in the group D, and the number of ~ 5 C cells in the groups A, B, C was markedly lower than that in the group D (P%0.01) . Also, there was very significant dif- ference in the number of 2C and ~ 5 C cells between group A and B or C (P%0.01) . It was conclud- ed that A-L tonic capsule could effectively inhibit the increase of DNA content of hepatocytes and im- prove the distribution of DNA content in rat hepatocarcinogenesis, especially in group A. Key words: hepatocellular carcinoma, experimental; DNA content; A-L tonic capsule

M e a s u r e m e n t of cell nuclear D N A con ten t , wh ich can di rec t ly reflect the prol i fera t ive and malig- nan t degree of cell dur ing carc inogenes is , was one of the useful me thods to de tec t t u m o r at present . A - L tonic capsule was a p repara t ion of compound prescr ip- t ion main ly made of astragalus rnembranaceus ( A M ) and ligustrum luciclum ( L L ) accord ing to the theory of T rad i t i ona l Chinese Medic ine " s t r e n g t h e n i n g the body resis tance to e l iminate pa thogen ic fac tors" . Pre- vious s tudies showed tha t A-I~ tonic medicine could enhance the i m m u n i t y of body c1'27. In the present s t u d y , d ie thy ln i t rosamine ( D E N A ) , one kind of car- c inogens , was used to induce expe r imen ta l hepatocel- lular carc inoma in rat . Dur ing this p rocess , the ra ts were fed wi th A - L tonic capsule using dif ferent m e t h o d s , and then the effects of A - L tonic capsule on D N A conten t in ra t expe r imen ta l hepa tocar - c inogenesis was inves t iga ted by using image analysis sys tem.

1 MATERIALS AND METHODS

1.1 Reagents and Doses D E N A . produc t of S igma ( U S A ) . 1 M solution

was f reshly made in a b r o w n bot t le . T h e ra ts were fed wi th D E N A by gas t rogavage one t ime a week wi th the dose of 70 m g / k g body weight every week EaT. A - L tonic capsule : provided by Dingxi pharmaceu t i ca l f ac to ry , Gansu . Suspens ion of A - L tonic capsule was f reshly m a d e , the ra ts were fed w i th the suspension by gas t rogavage two t imes a week wi th the dose of 15 .57 g / k g body weight every week E4~.

GUAN Yang, male, born in 1964, Lecturer

1.2 Animal Models Tota l ly 75 male SD ra t s , weighing 200--1-_20 g ,

purchased f rom Exper imen ta l An ima l Co. L t d , S h a n g h a i , were employed in this s tudy. F i f teen ra ts as no rma l control group were fed wi th c o m m o n rat diet and the remain ing 60 rats as expe r imen ta l group were admin i s te red wi th D E N A for to ta l 15 weeks. T h e expe r imen ta l group was fur ther divided into four subgroups inc luding: group A, the ra ts were previ- ously fed wi th A - L tonic capsule for 8 weeks and then D E N A added ; group B, D E N A and A - L tonic capsule were s imul taneous ly admin i s t e red ; group C , the ra ts were admin i s te red wi th D E N A for 7 weeks and then A - L tonic capsule added ; group D , the ra ts were only admin i s te red wi th D E N A . E x c e p t the ra ts of group A were killed at the 28 th w e e k , all ra t s of o ther g roups were killed at the 20 th week count ing f rom the beginning of exper iment . Liver t issues were f ixed by 4 % pa ra fo rma ldehyde and e m b e d d e d in paraff in. F o u r fam sect ions were cu t , cell nuclear D N A was s ta ined by modfied F e u l g e n ' s method . 1.3 Measurement of DNA Content by Automatic Image Analysis Technique

T J T Y - 3 0 0 au tomat ic image analysis sys tem was used t o measure D N A content . L igh t source was halogen lamp wi th the wave length of 560 n m and ob- jective magnif ica t ion of microscopy being 40 t imes. Gray-sca le of t issue gap on section was control led at 228 + 0 . 5 . T h e process of measu remen t was as fol- lows: the cells to be measured were selected under a mic roscope , signals were collected by CCD camera and input to compute r sys tem. T h e ra t io of in tegra t - ing optical dens i ty and dens i ty of nuclei were calculat- ed and D N A relat ive content was obta ined . T h e his- t og ram of D N A content d is t r ibut ion was made using

(;UAN Yang et al. A-I. Tonic Capsule on DNA Content in Hepatocarcinogenesis 13

D N A relat ive content as a abscissa and cell absolute f requency as a ordinate . As con t ro l , I ) N A content of 50 lymphocy te s was measured as s t andard 2 compo- nents (2C) ceils on the sect ions of each group. Abou t 350 nuclei of hepa tocytes or hepa toma cells were ran- domly measured on the sect ions of each group. The average was calculated and s ta t is t ical analysis was done.

2 R E S U L T S

D N A relat ive content of normal liver cells was 2. 38 and mainly composed of 2C cel ls , and there were also few 4C cells in normal liver tissue. In each exper imenta l g roup , the highest to lowest 1-)NA c o n -

tent were seen in the sequence of g roups I ) , B . C and A wi th the d i f ference of D N A conten t be ing m a r k e d - ly s ignif icance be tween group A or B or C and g roup I ) , and also be tween group A and group B or C ( P % 0. 01 , table 1 ). 1-)NA content d i s t r ibu t ion in the group A was very close to tha t of normal cont ro l group. H o w e v e r . the main cells in the group D were 4(" and ~ . ~ 5 C , 2(; cell was the minor i ty . T h e n u m b e r of 2C cells in the groups A , B, C was s ign i f ican t ly h igher than tha t in the group I) , and the n u m b e r of ~ 5 C cells in the group A , B, C was m a r k e d l y lower than tha t in the group D ( P ' ~ 0 . 01 ) . T h e di f fer - ences were also seen be tween group A and g roup B or C ( P ~ 0 . 0 1 , table 2) . T h e h i s tog ram of D N A con- tent d i s t r ibu t ion was shown in fig. 1.

150

125 g log

75

~ 5C

o 0

G'oup B G~oup A

,Ill,,. .,.I I,,,.. 2C 4C 6C 8C 2C 4C 6C 8C

GroJa C

�9 ,,!l!,,,,,, 2C 4C 6C 8C

DNA relative conter~t

Group D

..!, !l,,....,. 2C 4C 6C 8C 10C

Normal

I Ih. 2C 4C

Fig. 1 "['he histogram of DNA content distribution of normal and each experimental group

Table 1 D N A contents of normal and each exper imenta l group (2"-+- s )

Groups Relative DNA content

A 2 .92~0 . 907 "7' B 3.65-- 1.11" C 3.61-t-1.04" D 4..12+ 1. ,19

Normal Control 2 .38 • 0.691 �9 1)~0.01 as compared with group D (t- test) , /' P ~ 0 . 0 1 as compared with groups B or C (t-test)

Table 2 D N A content distr ibution of normal and each ex- perimental group

Groups Ploidy

2C ,IC ~>5C

A 43.58" = 52.71 3.57" " B 15.15" 70.00 1,,l. 85" C 1,1.63" 72.07 13.30" D 2 . 2 7 68.5I 29.22

Normal control 68.70 31.30 �9 P%0.01 as compared with group D (t-test) ~* P '~0 .01 as compared with group B or C (t-test)

3 DISCUSSION

D N A content in somat ic cell nuclei of various tis- sues is considerable ly c o n s t a n t , the average of this D N A conten t is very s imilar and twice to that of sperm cell and not varied with ind iv idua l , s ex , age and race, this k ind of cell is called 2C ceils. Except essential 2C cells, there are a small amoun t of ,IC

cells in some kinds of t i ssues , such as l iver , k idney and hear t ':s2. D N A content of cell nuclei is increased dur ing carcinogenesis . Compared with tha t of no rma l t i s sue , we can under s t and the varied range of D N A conten t in t u m o r cells, r ) N A content of ma l ignan t tu- mor cells was m a r k e d l y h igher (po lyp lo id or aneu- p loid) than that of normal t issue cells , the h i s t o g r a m of D N A content d is t r ibut ion became w i d e , the h igh peak was shif ted to the r ight '-52. Th i s p h e n o m e n o n was also d e m o n s t r a t e d by previous s tudies on h u m a n and e xpe r ime n t a l hepatocel lu lar ca rc inoma :~'r:j. T h e r e f o r e , obse rva t ion of var ia t ions of cell D N A con- tent and ploidy d is t r ibu t ion could help us to c o mpre - hend the prol i fera t ive and mal ignan t degree of cells du r ing carc inogenesis .

To unde r s t and the influence of A - L tonic cap- sule on D N A conten t in rat e xpe r ime n t a l hepa toca r - c inogenes is , in the present s t u d y , the ra t s were pre- v ious ly , s imul taneous ly and subsequen t ly fed wi th A - I. tonic capsule while they were admin i s t e red wi th D E N A , D N A content of all rat liver t i ssues was mea- sured using au tomat i c image analysis technique 20 weeks af te r admin i s t r a t ion of D E N A and c o m p a r e d wi th no rma l control . T h e resul ts showed tha t D N A conten t of group D , in which the ra ts were only ad- min i s t e red wi th D E N A , was m a r k e d l y h ighe r than tha t of no rma l cont ro l g r o u p , 4C and ~ 5 C cells were the ma jo r i t y in the group D. H o w e v e r , in the g roups A , B and C , in which the ra ts were a d m i n i s t e r e d w i th D E N A and A - I . tonic capsu le , D N A con ten t

(Continued on page 16)

16 Journal of Huazhong University of Science and Technology [Med Sci] 22 (1): 1,1-16, 2002

T h r o u g h o u t the s tudy period, there were no ad- verse events reported. The absorption and elimina- tion of both preparat ions were similar. Statistical analysis showed that there were no significant differ- ences among the main parameters , A U C ..... C .... and t .... ( P ~ > 0 . 0 5 ) , and the two formulat ions were bioe-

quivalent.

REFERENCES

1 ~-~ll~a.-~.l.-A-,-~,~ , $ ~ . 4f ' .~ .g , ' .~ .g- ,~@~, ' ]~ '~-~

(2) : 9,1 2 ~ , ~ - E . . ~ , t ! ~ @ ~ - a k ~ k ~ g ~ t o l ~ . ~ ) - ~ _ ~ .

~, IN ~b? #j _~ ,,~ & ~ ,~, 2000,] 9(2) :135 3 Gillis J C, Markham A. Irhe.~'~rtan: a review of its phar-

macodynamic and pharmacokinetics properties and thera- peutic use in the management of hypertension. Drugs, 1997,5,1(6) :885

4 Vachharaiani N N, Shyu W C, Chando T Jet al. Oral bioavailM)iIity and disposition characteristics of irbesar- tan, an angioten.sion antagonist, in healthy volunteers. J Clin Pharmacol, t.998,38 : 702

(Received Nov. 9, 2001)

(Continued from page 11 )

~g~, 2000,29(3):223 7 Fisher S J, Werb Z. The catabolism of extracellular ma-

trix components. In : I-{aral-son M A, Hassell(eds), Ex- tracellular matrix: a practical approach. Oxford: IRL Press, 1995. 260--287

8 Marteli M, Campama A, Bischof P. Secretion of matrix metalloproteinases by Human endometria[ cells in vitro. J Reprod Fertil, 1993,98:67

9 Freitas S, Mednri G, Le Nestoir E et al. Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium. Biolreprod, 1999,61(4):1070

10 Salamonsen L A, Kovacs G T, Findlay J K. Current concepts of the mechanisms of menstruation. Baillieres Best Pract Res ('lin Obstet Gynaecol, 1999,13(2):161

11 Vincent A J, Zhang J, Ostor A et al. Matrix metrallo- proteinase-1 and 3 and mast cells are present in the en-

dometrium of women single progestin-only contracep- tives. Hum reprod, 2000, 15(1)=123

~..~, 1995,30(9) :516 ~3 -~,~.a~t,~. ~ - g ~ z Y ~ ~

~fl~J{ak..~.N,.ikgj~J,~@N.'/l~. ~ ' L g ~ # g q t ~ , ~ , 1993, 30(9) : 526

~.~/~@Nv~,~@~4e,@tg. _~Jf. Nir~_-a ~ , 1993,13(2): 105

31(9):523 16 Fisher C, gilberton-Beadlig S, Powers E A et al. later-

stitial collagenase is required for angiogenesis in virr-_ Dev Biol, 1994,162:499

(Received June 5, 2001)

(Continued from page 13) and the n u m b e r of ~ 5 C cells were significantly lower than that in the group I) (bo th P % 0 . 01) . D N A content of group A in which the rats were preven- tively fed with A - L tonic capsule was the lowest a- mong the groups A , B, C and the difference was sig- nificant between group A and group B or C ( P % 0 . 01) . 2C and 4C cells were predominant and ~ 5 C cells were few in the group A. Therefore , A-L tonic capsule could effectively inhibit the increase of D N A content of liver cells and improve the dis tr ibut ion of D N A ploidy in rat hepatocarcinogenesis , especially in the group A.

About the mechanism of A - L runic capsule in- hibi t ing the increase of DNA content of liver cells, we deduced that it might be associated with the im- m u n o e n h a n c e m e n t of A-L tonic capsule. In other ex- pe r imen t , we noticed tha~ A- I . tonic capsule could ef- fectively enhance N K cell act ivi ty , raise the serum concentra t ions of IL-2 and T N F , thus inhibi t ing hepatocarcinogenesis and development of hepatocellu-

lar carcinoma.

REFERENCES

1 - /4 . .~ ,Hersh E M , ~ ' ~ : ~ . . t s 1 7 6

2 Yan S, Evan M H, Siu-Leung I. et al . Preliminary ob- servations on the effects of the Chinese medicinal herbs Astragalus membranaceus and Ligustrum lucidum on lymphocyte blastogenic responses. J Biol Resp Modif, 1983,2:227

3R:~. ~ p ~ . ~ . , ~ , 1992,21:113 4 - / . l , - ~ , - L . . ~ - , I - ~ . . ~ g . ~ . : . & N , ~ N _ 4 z k , 1987.2.15

D N A 4 t ~ N j ~ , ~ . . ~@-~-"Y-'~-,'k-.,g-, 1991,5=98

~. ,~ , 1992,2:85 (Received Sept. 21, 2001)