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Sensitive detection of growth stimulating tumour promoters in perifused primary rat hepatocyte cultures
Pergamon
Toxicology in Vitro 1 I (1997) 543-547
Sensitive Detection of Growth Stimulating Tumour Promoters in Perifused Primary Rat Hepatocyte Cultures S. KLEIN Physiologisch-chemisches
Institut
and
R. GEBHARDT*
der Universitiit
Tiibingen,
D-72076
Tiibingen,
Germany
Abstract-Comparison of stationary cultivation with discrete changes of the medium with pcrifusion. where the hepatocytes are cultivated under a continuous flow of medium showed an enhanced sensitivity of perifused hepatocytes towards the mitogen cyproterone acetate (CPA) and the potential tumour promoters lindane, 4-chloro-I-naphthol, dieldrin and clofibrate. Addition of these substrates significantly stimulated DNA synthesis in the perifusion cultures while stationary cultures remained dormant or reacted in a less pronounced manner. In contrast, I-methyl-3-nitro-I-nitroso-guanidine (MNNG) at 5 PM showed no effect on the incorporation of bromodeoxyuridine (BrdU) in both systems. The increase in sensitivity may be due to physical (establishment of steady-state concentrations) and physiological advantages (enhanced biotransformation, intact signal transduction pathways) of the perifusion culture. These studies demonstrate that perifusion is more suitable than stationary rat hepatocyte cultures with respect to the detection of growth stimulatory effects of tumour promoters, as previously reported for growth inhibitory effects of some genotoxic carcinogens. Therefore, the p&fusion may considerably aid in discriminating between growth inhibitory and growth stimulatory effects of environmental chemicals. 0 1997 Ekeuier
Science
Ltd
Abbreviations: BrdU = bromodeoxyuridine; CPA = cyproterone acetate; EGF = epidermal growth factor; GF = growth factor; LDH = lactate dehydrogenase; Ll = labelling index; MNNG = I-methyl-3nitro-I-nitroso-guanidine.
INTRODUCTION Among
carcinogens,
genotoxic
chemicals
often
cause
in rodent hepatocytes (Farber el al., 1977; Moore and Kitagawa, 1986), while tumour promoters frequently induce a non-toxic transitory growth response (Murray et al., 1989; Schulte-Hermann et al., 1980; Thottassery and Yarbrough, 1991). In uiuo, the latter compounds, including cyproterone acetate, nafenopin, hexachlorocyclohexane, mirex and various others, may induce adaptive liver growth comprising both hyperplasia and hypertrophy (Levin et al., 1977; Schulte-Hermann, 1974 and 1978; Schulte-Hermann et al., 1981; Yarbrough et al., 1992). Primary cultures of rat hepatocytes have proved useful for studies on the modulation of hepatocyte growth (McGowan, 1986; Michalopoulos, 1990; Nakamura et al., 1988; Volk et al., 1995) and have been used for determining the potency of chemicals to either inhibit or stimulate hepatocyte growth (Gebhardt and Fischer, 1995; Novicki ef aI., 1985; Yusof and Edwards, 1990). H >wever, many tumour promoters known to stimulate liver growth in uioo mitoinhibition
*Author for correspondence Institut D-72076
at: Physiologisch-chemisches der Universitiit, Hoppe-Seyler-Strasse Tiibingen, Germany.
0887-2333/97/%17.00 + 0.00 0 SSDI 0887-2333(97)00063-5
1997 Elsevier Science
4,
such as mirex show no stimulatory action - not even as co-mitogens in normal rat hepatocyte cultures. Recently, we have reported on striking differences in the mitoinhibitory potential of several genotoxic chemicals depending on whether stationary culture conditions with discrete changes of the medium or a perifusion system allowing continuous exchange of medium was used (Gebhardt and Fischer, 1995). In the perifusion system mitoinhibition was much more pronounced and the hepatocytes were more sensitive to lower concentrations of the genotoxic carcinogens (Gebhardt and Fischer, 1995). Therefore, we wondered whether the perifusion system would also prove superior in detecting growth stimulatory effects of tumour promoters. In the present study we report on differences in the growth stimulatory potency of cyproterone acetate and some other tumour promoters in stationary and perifused rat hepatocyte cultures. MATERIALS AND METHODS
Hepatocyte culture and treatment Adult male Sprague-Dawley rats (220-280 g) were used in these experiments. After isolation the hepatocytes were seeded on collagen-coated glass supports lying in the culture chambers of the
Ltd. AI1 rights
reserved.
Printed
in Great
Britain
S. Klein and R. Gebhardt
544
perifusion (Gebhardt started 24 tion: half stationary connected
system and cultured for 24 hr as described and Fischer, 1995). The comparison hr after isolation and stationary cultivaof the chambers were cultured under conditions, while the other half was with the perifusion system, described in
detail elsewhere (cf. Gebhardt, 1994; Gebhardt and Mecke, 1979). The treatment with substrates of interest started 24 hr after plating and lasted for additional 48 hr. The chemicals that were tested for their interference with the growth of the hepatocytes, namely CPA, lindane, 4-chloro- I -naphthol, dieldrin, clofibrate and MNNG, were added to the culture medium in the concentrations indicated in the text, legends to figures and in the table. Some experiments were carried out in the presence of insulin and EGF, their concentrations are also indicated in the text, legends to figures and in the table. The perifusion system contained a total of 60 ml culture medium and was run at 10 ml/hr in the closed mode. In the stationary cultures, medium (10 ml) was changed
every 24 hr. During the last 24 hr before termination of the experiment 100 pg BrdU/chamber was added to label the newly-synthesized DNA. Cytotoxicity was determined using leakage of lactate dehydrogenase (LDH) as described by Gebhardt et ai. (1996). Determination of‘ DNA synthesis
For immunocytochemical detection of BrdUlabelled hepatocytes the cultures were rinsed with buffered saline and fixed with ice-cold 3.5% paraformaldehyde for 25 min. Further processing and immunocytochemical staining for BrdU-labelled nuclei was carried out as described elsewhere (Volk et al., 1995). To compare stationary cultivation and perifusion the LI was used. The LI represents the percentage of BrdU-labelled nuclei per total number of nuclei counted on each support (10 microscopic fields per support with 7k-100 cells per field) (Novicki et al., 1985; Volk et al., 1995). The values shown represent means i SD calculated from three supports pooled from the number of experiments stated in the figure legends and table. Student’s t-test was used for statistical evaluation.
by CPA at 10 pM as we11 as at 35 FM to a much greater extent. In the case of lindane, 4-chloro-I-naphthol, dieldrin and clofibrate the enhanced sensitivity of perifused hepatocytes was as well pronounced: in the presence of IO nM insulin and 0.67 PM EGF addition of 1 pM lindane significantly increased the LI in perifused cultures, while stationary cultures remained dormant or even showed a slight tendency to decrease the LI as for clofibrate at low concentrations of growth factors (Table I). Interestingly, even raising the concentrations to IO PM had no effect in the stationary cultures, while the perifused hepatocytes were stimulated to a similar extent. Moreover, in the perifusion system addition of lindane (1 or 10 PM) stimulated the LI beyond a LI of approximately 35%, that can be reached with growth factors (Gebhardt and Fischer, 1995) indicating its true action as a co-mitogen (Table 1). No such effect could be observed in stationary cultures. Similar differences were found for the growth-promoting effects of 4-chloro-I-naphthol, dieldrin and clofibrate, while MNNG at 5 pM showed no effect on the incorporation of BrdU (Table 1). Based on morphological criteria and the LDHleakage assay, no signs of cytotoxicity were observed for the highest concentrations of all compounds used in this study. Moreover, with the exception of CPA (no solubility above 35 pM) and 4-chloro-1-naphthol
(starting at 100 pM) cytotoxicity did not even start at 250 PM.
of all compounds
DISCUSSION
In agreement with the results of Parzefall et al. (1985 and 1989) that CPA can act as a co-mitogen,
60
c
_
RESULTS In the presence of growth promoting factors (insulin and EGF at concentrations of IO nM and 0.67 PM, respectively) addition of CPA as low as 5 pM
tended to increase the LI in perifused cultures (1.7-fold control), while stationary cultures remained dormant (Fig. 1). In the case of 10 pM and 35 PM CPA the difference between hepatocytes cultured in both systems was even more pronounced. The comparison of CPA-stimulated LI in perifusion and stationary culture in the presence of various concentrations of the growth factors Insulin and EGF (Fig. 2) indicated that in the presence of these growth factors perifused hepatocytes were stimulated
IO
Cyproteroneacetate(PM) Fig. 1. Comparison of CPA-stimulated BrdU-labelling indices in perifusion (0-O) and stationary culture (m-m). Hepatocytes cultured in the presence of insulin (10 nM)and EGF (0.67 PM = 4 ng/ml) were exposed to various concentrations of CPA. Data represent means f SD from three supports pooled from four different experiments. (a) different from respective control; P < 0.05: and (b) different from respective control; P < 0.001.
Tumour
promoters
in perifused
hepatocytes
IO
lo()nM
1.67 PM
5 IIM 0.34 PM
Insulin EGF
Fig. 2. Comparison of CPA-stimulated BrdU-labelling indices in perifusion and stationary culture. Hepatocytes were exposed to various concentrations of insulin, EGF and CPA. Data represent means i SD from three supports pooled from three different experiments. (a) statistically different from respective control; P < 0.03: (b) statistically different from respective control; P < 0.001: and (c) not significant.
we found a concentration-dependent rise of the LI stationary cultures of hepatocytes exposed to CPA concentrations higher than 10 FM. However, demonstrated in this study the CPA response
Table I.
Influence of lindane, 4-chloro-I-napbthol, stationary
in
hepatocytes cultured in the perifusion system was more pronounced, indicating the suitability of the perifusion system concerning studies on the effects of tumour promoters. These differences were significant
at
as of
dieldrin, clofibrate and MMNG
culture in the presence of growth
on the LI of hepatocytes maintained factors (GF) at low and high concentrations
Stationary
Control Lindane l I’M IO pLM 4-Chloro-I-naphthol
1w IO pLM Dieldrin
1 W’ IO pLM Clofibrate
1 PM IO PM MNNG 5 PM
cultivation
in perifusion
P&fusion
GF Iowa
GF hlghb
GF low”
LI
Ll
LI
LI
9.7 * 1.0
33.4 f 0.9
15.0 * 5.2
34.0 + 1.8
8.0 + 0.6’ 8.7 f 0.7d
33.2 + 2. Id 32.2 F 1.5”
23.7 + 3.9” 24.2 k 1.0’
43.2 i 2.9 44.7 * 3.3’
8.3 i 0.4’ 10.0 * 0.94
n.d. 31.8 f I.Zd
18.6 & 0.9’ 18.9 f 2 5’
n.d. 43.2 f I.9
8.9 f 0.84 8.5 + 0.8”
32.0 f O.Yd 34.7 * 1.9”
27.9 + 1.5’ 30.5 * 3.1”
46.3 + 1.5’ 49.1 * 3.5
7.6 f 0.6e 7.9 f 0.59
32.0 * 1.4d 32.9 f l.35
25.1 f 0.9’ 27.6 f 0.6’
40.9 f 1.6 43.2 + 3.9
n.d.
34.5 * 1.5”
n.d.
35.6 + I.@
LI in the absence of GF: stationary cultivation: 1.5 5 0.7; perifusion: 7.3 + 1.2. “Addition of IO nM insulin and 0.67 PM (4 r&ml) EGF to culture medium. “Addition of 100 nM insulin and 1.67 PM (10 “g/ml) EGF to culture medium. ‘Data represent means + SD from a total of six different experiments. dNot significantly different from controls. ‘Statistically significant from respective control; P < 0.001. ‘n.d. = not determined. gDifferent from respective control: P < 0.03.
GF high”
and
546
S. Klein and R. Gebhardt
despite slightly higher LI values in non-treated controls of perifused hepatocytes compared with stationary cultures. This increased growth response (among other things, probably due to optimized nutrient supply) was already observed in previous studies and was discussed therein (Gebhardt and Fischer. 1995). With respect to CPA, it is interesting to note that this compound was found to produce DNA adducts (Neumann et al., 1992) and thus must also be classified as a genotoxic agent, as also demonstrated by Martelli er a/. (1995). However. the fact that stimulation of growth rather than mito-inhibition [as found for other genotoxic compounds (Gebhardt and Fischer, l995)] is found in cultured hepatocytes indicates that CPA has a very high co-mitogenic potential explaining its high tumour promoting effect observed in viva (Schulte-Hermann et cd.. 1981). The advantage of perifused hepatocytes was even more obvious from studies on the effects of the potential tumour promoters lindane. 4-chloro- I naphthol, dieldrin and clofibrate. In contrast to the co-mitogen CPA, DNA stimulation by these compounds could only be found in perifused hepatocytes, while stationary cultures showed no effect up to 10 pM. Similar differences between the two culture systems were found in experiments investigating growth inhibitory effects of genotoxic carcinogens (Gebhardt and Fischer, 1995). These results demonstrate an enhanced sensitivity of perifused hepatocytes with respect to the interference of chemicals with hepatocellular growth regulation in general. The enhanced sensitivity may be due to several reasons: (i) The shallow gradients of infused compounds in the closed perifusion mode, corresponding to nearly steady-state conditions, may prolong the period of exposure of the hepatocytes to these compounds even at low concentrations thus resulting in a higher efficacy of these molecules. (ii) Perifused hepatocytes, in general, maintain a better metabolic performance (Gebhardt, 1994; Gebhardt et al.. 1996). (iii) Likewise, this may also hold for the efficiency of signal transduction pathways regulating cell growth. Current research is directed to investigate the latter possibilities. Besides mitostimulation, tumour promoters are known to inhibit apoptosis in vice (Stinchcombe et al., 1995) and in vitro (Oberhammer and Qin, 1995). It is possible that perifusion will be a useful aid in future work on elucidating the relative contribution of apoptosis and mitostimulation to the action of tumour promoters and other xenobiotics.
REFERENCES
Farber E., Solt D., Cameron R., Laishes B., Ogawa K. and Medline A. (1977) Newer insights into the pathogenesis of liver cancer. American Journal af Pathology 89, 477-482. Gebhardt R. (1994) Improved drug metabolizing capacity of hepatocytes co-cultured with epithelial cells and maintained in a perifusion system. In Alternatives to Animal Testing. Edited by C. Reinhard. pp. 141-146. Verlag Chemie, Weinheim. Gebhardt R. and Fischer S. (1995) Enhanced sensitivity of perifused primary rat hepatocytes to mitogens and growth modulation by carcinogens. ToxicoIogJl in Vitro 9, 44545
I.
Gebhardt R. and Mecke D. (1979) Perifused monolayer cultures of rat hepatocytes as an improved in vitro system for studies on ureogenesis. E.uperimental Cell Research 124, 349-359.
Gebhardt R., Wegner H. and Alber J. (1996) Perifusion of cocultured hepatocytes: optimization of studies on drug metabolism and cytotoxicity in vitro. CeN Biology and To.+ology
12, 57-68.
Levin W. G., Ord M. G. and Stocken L. A. (1977) Some biochemical changes associated with nafenopin-induced liver growth in the rat. Biochemical Pharmacology 26, 939-942.
McGowan J. A. (1986) Hepatocyte proliferation in culture. In Research in Isolated and Cultured Hepatocytes. Edited by A. Guillouzo and C. Guguen-Guillouzo. pp. 13-38. Inserm, Paris, and John Libbey Eurotext, Montrouge. Martelli A., Mattioli F., Fazio S.. Andrae U. and Brambilla G. (1995) DNA repair synthesis and DNA fragmentation m primary cultures of human and rat hepatocytes exposed to cyproterone acetate. Curcinogenesis 16, 1265-1269. Michalopoulos G. K. (1990) Lever regeneration: molecular mechanisms of growth control. FASEB Journal 4, 176-187.
Moore M. A. and Kitagawa T. (I 986) Hepatocarcinogenesis in the rat: the effect of promoters and carcinogens in vivo and in vitro. International Review of Cvtology 101, 125-173. Murray A. W.. Edwards A. M. and Hii C. S. T. (1989) Tumour promotion: biology and molecular mechanisms. In Carcinogenesis and Mutagenesis. Edited by P. L. Grover and C. S. Cooper. pp. 133-157. Springer, Berlin. Nakamura T., Arakaki R. and Ichihara A. (1988) Interleukin-I is a potent growth inhibitor of adult rat hepatocytes in primary culture. E.uperimenfaI Cell Research
179, 488497.
Neumann I., Thierau D., Andrae U., Greim H. and Schwarz L. R. (1992) Cyproterone acetate induces DNA damage in cultured rat hepatocytes and preferentially stimulates DNA synthesis in gamma-glutamyltranspeptidase-positive cells. Carcinogenesis 13, 373-378. Novicki D. L., Rosenberg M. R. and Michalopoulos G. (1985) Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes. Cancer Research 45, 337-344. Oberhammer F. A. and Qin H. M. (1995) Effect of three tumour promoters on the stability of hepatocyte cultures and apoptosis after transforming growth factor-beta 1. Carcinogenesis 16, 1363-l 37 I. Parzefall W., Galle P. R. and Schulte-Hermann R. (1985) Effect of calf and rat serum on the induction of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes by cyproterone acetate and epidermal growth factor. In Vitro Cellular and Developmental Biology 21, 665-673.
Acknowledgements-This
work was supported
by the
Deutsche Forschungsgemeinschaft (Forschergruppe Biochemische Toxikologie).
Parzefall W., Monschau P. and Schulte-Hermann R. (1989) Induction by cyproterone acetate of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes in serum free medium. Archives af Toxicology 63,45W61. Schulte-Hermann R. (1974) Induction of liver growth by
Tumour
promoters
xenobiotic compounds and other stimuli. Crirical in Toxicology 3, 97- 158. Schulte-Hermann R. (1978) proliferation
Two-stage
control
in perifused hepatocytes
Reoiews of
cell
induced in rat liver by hexachlorocyclohex-
ane. Cancer Research 37, 16171. Schulte-Hermann R.. Hoffmann V., Parzefall W., Kallenbath M., Gerhardt A. and Schuppler J. (1980) Adaptive responses of rat liver to the gestagen and anti-androgen cyproterone acetate and other inducers II. Induction of growth. Chemico-Biological Inrerocrions 31, 287-300. Schulte-Hermann R., Ohde G., Schuppler J. and Timmermann-Trosiener I. (1981) Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with the tumour promoters phenobarbital, steroid compounds. and hexachlorocyclohexane, nafenopin. Cancer Research 41, 2556-2562. Stinchcombe S.. Buchmann A., Bock K. W. and Schwarz M. (1995) Inhibition of apoptosis during 2.3.7,8-tetra-
547
chlorodibenzo-p-dioxin-mediated tumour rat liver. Corcinogenesis 16, 1271-1275. Thottassery J. V. and Yarbrough of glucocorticoid receptors
promotion
in
J. D. (1991) Regulation during adaptive liver
growth. American Journal of Physiology 260, G603xI609. Volk A., Michalopoulos G.. Weidner M. and Gebhardt R. (1995) Different proliferative response of periportal and pericentral rat hepatocytes to hepatocyte growth factor. Biochemicul and Biophysical Research Communicotiom 207, 578-584. Yarbrough J. D., Grimley J. M. and Thottassery J. V. (1992) Mirex-induced adaptive liver growth in rats subjected to thyroidectomy. Heporology IS, 923-927. Yusof Y. A. M. and Edwards A. M. (1990) Stimulation of DNA synthesis in primary rat hepato-cyte cultures by liver tumour promoters: interaction with other growth factors. Carcinogenesis I I, 761 770.
Toxicology in Vitro 1 I (1997) 543-547
Sensitive Detection of Growth Stimulating Tumour Promoters in Perifused Primary Rat Hepatocyte Cultures S. KLEIN Physiologisch-chemisches
Institut
and
R. GEBHARDT*
der Universitiit
Tiibingen,
D-72076
Tiibingen,
Germany
Abstract-Comparison of stationary cultivation with discrete changes of the medium with pcrifusion. where the hepatocytes are cultivated under a continuous flow of medium showed an enhanced sensitivity of perifused hepatocytes towards the mitogen cyproterone acetate (CPA) and the potential tumour promoters lindane, 4-chloro-I-naphthol, dieldrin and clofibrate. Addition of these substrates significantly stimulated DNA synthesis in the perifusion cultures while stationary cultures remained dormant or reacted in a less pronounced manner. In contrast, I-methyl-3-nitro-I-nitroso-guanidine (MNNG) at 5 PM showed no effect on the incorporation of bromodeoxyuridine (BrdU) in both systems. The increase in sensitivity may be due to physical (establishment of steady-state concentrations) and physiological advantages (enhanced biotransformation, intact signal transduction pathways) of the perifusion culture. These studies demonstrate that perifusion is more suitable than stationary rat hepatocyte cultures with respect to the detection of growth stimulatory effects of tumour promoters, as previously reported for growth inhibitory effects of some genotoxic carcinogens. Therefore, the p&fusion may considerably aid in discriminating between growth inhibitory and growth stimulatory effects of environmental chemicals. 0 1997 Ekeuier
Science
Ltd
Abbreviations: BrdU = bromodeoxyuridine; CPA = cyproterone acetate; EGF = epidermal growth factor; GF = growth factor; LDH = lactate dehydrogenase; Ll = labelling index; MNNG = I-methyl-3nitro-I-nitroso-guanidine.
INTRODUCTION Among
carcinogens,
genotoxic
chemicals
often
cause
in rodent hepatocytes (Farber el al., 1977; Moore and Kitagawa, 1986), while tumour promoters frequently induce a non-toxic transitory growth response (Murray et al., 1989; Schulte-Hermann et al., 1980; Thottassery and Yarbrough, 1991). In uiuo, the latter compounds, including cyproterone acetate, nafenopin, hexachlorocyclohexane, mirex and various others, may induce adaptive liver growth comprising both hyperplasia and hypertrophy (Levin et al., 1977; Schulte-Hermann, 1974 and 1978; Schulte-Hermann et al., 1981; Yarbrough et al., 1992). Primary cultures of rat hepatocytes have proved useful for studies on the modulation of hepatocyte growth (McGowan, 1986; Michalopoulos, 1990; Nakamura et al., 1988; Volk et al., 1995) and have been used for determining the potency of chemicals to either inhibit or stimulate hepatocyte growth (Gebhardt and Fischer, 1995; Novicki ef aI., 1985; Yusof and Edwards, 1990). H >wever, many tumour promoters known to stimulate liver growth in uioo mitoinhibition
*Author for correspondence Institut D-72076
at: Physiologisch-chemisches der Universitiit, Hoppe-Seyler-Strasse Tiibingen, Germany.
0887-2333/97/%17.00 + 0.00 0 SSDI 0887-2333(97)00063-5
1997 Elsevier Science
4,
such as mirex show no stimulatory action - not even as co-mitogens in normal rat hepatocyte cultures. Recently, we have reported on striking differences in the mitoinhibitory potential of several genotoxic chemicals depending on whether stationary culture conditions with discrete changes of the medium or a perifusion system allowing continuous exchange of medium was used (Gebhardt and Fischer, 1995). In the perifusion system mitoinhibition was much more pronounced and the hepatocytes were more sensitive to lower concentrations of the genotoxic carcinogens (Gebhardt and Fischer, 1995). Therefore, we wondered whether the perifusion system would also prove superior in detecting growth stimulatory effects of tumour promoters. In the present study we report on differences in the growth stimulatory potency of cyproterone acetate and some other tumour promoters in stationary and perifused rat hepatocyte cultures. MATERIALS AND METHODS
Hepatocyte culture and treatment Adult male Sprague-Dawley rats (220-280 g) were used in these experiments. After isolation the hepatocytes were seeded on collagen-coated glass supports lying in the culture chambers of the
Ltd. AI1 rights
reserved.
Printed
in Great
Britain
S. Klein and R. Gebhardt
544
perifusion (Gebhardt started 24 tion: half stationary connected
system and cultured for 24 hr as described and Fischer, 1995). The comparison hr after isolation and stationary cultivaof the chambers were cultured under conditions, while the other half was with the perifusion system, described in
detail elsewhere (cf. Gebhardt, 1994; Gebhardt and Mecke, 1979). The treatment with substrates of interest started 24 hr after plating and lasted for additional 48 hr. The chemicals that were tested for their interference with the growth of the hepatocytes, namely CPA, lindane, 4-chloro- I -naphthol, dieldrin, clofibrate and MNNG, were added to the culture medium in the concentrations indicated in the text, legends to figures and in the table. Some experiments were carried out in the presence of insulin and EGF, their concentrations are also indicated in the text, legends to figures and in the table. The perifusion system contained a total of 60 ml culture medium and was run at 10 ml/hr in the closed mode. In the stationary cultures, medium (10 ml) was changed
every 24 hr. During the last 24 hr before termination of the experiment 100 pg BrdU/chamber was added to label the newly-synthesized DNA. Cytotoxicity was determined using leakage of lactate dehydrogenase (LDH) as described by Gebhardt et ai. (1996). Determination of‘ DNA synthesis
For immunocytochemical detection of BrdUlabelled hepatocytes the cultures were rinsed with buffered saline and fixed with ice-cold 3.5% paraformaldehyde for 25 min. Further processing and immunocytochemical staining for BrdU-labelled nuclei was carried out as described elsewhere (Volk et al., 1995). To compare stationary cultivation and perifusion the LI was used. The LI represents the percentage of BrdU-labelled nuclei per total number of nuclei counted on each support (10 microscopic fields per support with 7k-100 cells per field) (Novicki et al., 1985; Volk et al., 1995). The values shown represent means i SD calculated from three supports pooled from the number of experiments stated in the figure legends and table. Student’s t-test was used for statistical evaluation.
by CPA at 10 pM as we11 as at 35 FM to a much greater extent. In the case of lindane, 4-chloro-I-naphthol, dieldrin and clofibrate the enhanced sensitivity of perifused hepatocytes was as well pronounced: in the presence of IO nM insulin and 0.67 PM EGF addition of 1 pM lindane significantly increased the LI in perifused cultures, while stationary cultures remained dormant or even showed a slight tendency to decrease the LI as for clofibrate at low concentrations of growth factors (Table I). Interestingly, even raising the concentrations to IO PM had no effect in the stationary cultures, while the perifused hepatocytes were stimulated to a similar extent. Moreover, in the perifusion system addition of lindane (1 or 10 PM) stimulated the LI beyond a LI of approximately 35%, that can be reached with growth factors (Gebhardt and Fischer, 1995) indicating its true action as a co-mitogen (Table 1). No such effect could be observed in stationary cultures. Similar differences were found for the growth-promoting effects of 4-chloro-I-naphthol, dieldrin and clofibrate, while MNNG at 5 pM showed no effect on the incorporation of BrdU (Table 1). Based on morphological criteria and the LDHleakage assay, no signs of cytotoxicity were observed for the highest concentrations of all compounds used in this study. Moreover, with the exception of CPA (no solubility above 35 pM) and 4-chloro-1-naphthol
(starting at 100 pM) cytotoxicity did not even start at 250 PM.
of all compounds
DISCUSSION
In agreement with the results of Parzefall et al. (1985 and 1989) that CPA can act as a co-mitogen,
60
c
_
RESULTS In the presence of growth promoting factors (insulin and EGF at concentrations of IO nM and 0.67 PM, respectively) addition of CPA as low as 5 pM
tended to increase the LI in perifused cultures (1.7-fold control), while stationary cultures remained dormant (Fig. 1). In the case of 10 pM and 35 PM CPA the difference between hepatocytes cultured in both systems was even more pronounced. The comparison of CPA-stimulated LI in perifusion and stationary culture in the presence of various concentrations of the growth factors Insulin and EGF (Fig. 2) indicated that in the presence of these growth factors perifused hepatocytes were stimulated
IO
Cyproteroneacetate(PM) Fig. 1. Comparison of CPA-stimulated BrdU-labelling indices in perifusion (0-O) and stationary culture (m-m). Hepatocytes cultured in the presence of insulin (10 nM)and EGF (0.67 PM = 4 ng/ml) were exposed to various concentrations of CPA. Data represent means f SD from three supports pooled from four different experiments. (a) different from respective control; P < 0.05: and (b) different from respective control; P < 0.001.
Tumour
promoters
in perifused
hepatocytes
IO
lo()nM
1.67 PM
5 IIM 0.34 PM
Insulin EGF
Fig. 2. Comparison of CPA-stimulated BrdU-labelling indices in perifusion and stationary culture. Hepatocytes were exposed to various concentrations of insulin, EGF and CPA. Data represent means i SD from three supports pooled from three different experiments. (a) statistically different from respective control; P < 0.03: (b) statistically different from respective control; P < 0.001: and (c) not significant.
we found a concentration-dependent rise of the LI stationary cultures of hepatocytes exposed to CPA concentrations higher than 10 FM. However, demonstrated in this study the CPA response
Table I.
Influence of lindane, 4-chloro-I-napbthol, stationary
in
hepatocytes cultured in the perifusion system was more pronounced, indicating the suitability of the perifusion system concerning studies on the effects of tumour promoters. These differences were significant
at
as of
dieldrin, clofibrate and MMNG
culture in the presence of growth
on the LI of hepatocytes maintained factors (GF) at low and high concentrations
Stationary
Control Lindane l I’M IO pLM 4-Chloro-I-naphthol
1w IO pLM Dieldrin
1 W’ IO pLM Clofibrate
1 PM IO PM MNNG 5 PM
cultivation
in perifusion
P&fusion
GF Iowa
GF hlghb
GF low”
LI
Ll
LI
LI
9.7 * 1.0
33.4 f 0.9
15.0 * 5.2
34.0 + 1.8
8.0 + 0.6’ 8.7 f 0.7d
33.2 + 2. Id 32.2 F 1.5”
23.7 + 3.9” 24.2 k 1.0’
43.2 i 2.9 44.7 * 3.3’
8.3 i 0.4’ 10.0 * 0.94
n.d. 31.8 f I.Zd
18.6 & 0.9’ 18.9 f 2 5’
n.d. 43.2 f I.9
8.9 f 0.84 8.5 + 0.8”
32.0 f O.Yd 34.7 * 1.9”
27.9 + 1.5’ 30.5 * 3.1”
46.3 + 1.5’ 49.1 * 3.5
7.6 f 0.6e 7.9 f 0.59
32.0 * 1.4d 32.9 f l.35
25.1 f 0.9’ 27.6 f 0.6’
40.9 f 1.6 43.2 + 3.9
n.d.
34.5 * 1.5”
n.d.
35.6 + I.@
LI in the absence of GF: stationary cultivation: 1.5 5 0.7; perifusion: 7.3 + 1.2. “Addition of IO nM insulin and 0.67 PM (4 r&ml) EGF to culture medium. “Addition of 100 nM insulin and 1.67 PM (10 “g/ml) EGF to culture medium. ‘Data represent means + SD from a total of six different experiments. dNot significantly different from controls. ‘Statistically significant from respective control; P < 0.001. ‘n.d. = not determined. gDifferent from respective control: P < 0.03.
GF high”
and
546
S. Klein and R. Gebhardt
despite slightly higher LI values in non-treated controls of perifused hepatocytes compared with stationary cultures. This increased growth response (among other things, probably due to optimized nutrient supply) was already observed in previous studies and was discussed therein (Gebhardt and Fischer. 1995). With respect to CPA, it is interesting to note that this compound was found to produce DNA adducts (Neumann et al., 1992) and thus must also be classified as a genotoxic agent, as also demonstrated by Martelli er a/. (1995). However. the fact that stimulation of growth rather than mito-inhibition [as found for other genotoxic compounds (Gebhardt and Fischer, l995)] is found in cultured hepatocytes indicates that CPA has a very high co-mitogenic potential explaining its high tumour promoting effect observed in viva (Schulte-Hermann et cd.. 1981). The advantage of perifused hepatocytes was even more obvious from studies on the effects of the potential tumour promoters lindane. 4-chloro- I naphthol, dieldrin and clofibrate. In contrast to the co-mitogen CPA, DNA stimulation by these compounds could only be found in perifused hepatocytes, while stationary cultures showed no effect up to 10 pM. Similar differences between the two culture systems were found in experiments investigating growth inhibitory effects of genotoxic carcinogens (Gebhardt and Fischer, 1995). These results demonstrate an enhanced sensitivity of perifused hepatocytes with respect to the interference of chemicals with hepatocellular growth regulation in general. The enhanced sensitivity may be due to several reasons: (i) The shallow gradients of infused compounds in the closed perifusion mode, corresponding to nearly steady-state conditions, may prolong the period of exposure of the hepatocytes to these compounds even at low concentrations thus resulting in a higher efficacy of these molecules. (ii) Perifused hepatocytes, in general, maintain a better metabolic performance (Gebhardt, 1994; Gebhardt et al.. 1996). (iii) Likewise, this may also hold for the efficiency of signal transduction pathways regulating cell growth. Current research is directed to investigate the latter possibilities. Besides mitostimulation, tumour promoters are known to inhibit apoptosis in vice (Stinchcombe et al., 1995) and in vitro (Oberhammer and Qin, 1995). It is possible that perifusion will be a useful aid in future work on elucidating the relative contribution of apoptosis and mitostimulation to the action of tumour promoters and other xenobiotics.
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by the
Deutsche Forschungsgemeinschaft (Forschergruppe Biochemische Toxikologie).
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