Role of Extracellular Zinc and Copper on Metallothionein Regulation in Cultured Rat Hepatocytes

JUAN HIDALGO, ALLEN DINGMAN AND JUSTINE S. GARVEY Department of Biology, Syracuse University, Syracuse, New York 13244

Cellular and extracellular metallothionein contents of rat hepatocytes cultured in the presence of albumin (30 pmoVL) with zinc (1,10,50 and 100 pmol/L), copper (1, 10 and 50 pmol/L), zinc and copper (1, 10 and 50 pmol/L of each metal) or no metals in the culture medium have been measured by radioimmunoassay. Cellular metallothionein levels increased steadily with culture time regardless of the metal treatment and showed little dependence (only a twofold increase) on extracellular zinc or copper at all metal concentrations and times (up to 3 days) studied. In contrast, the presence of both metals simultaneously in the culture medium strongly increased cellular metallothionein contents, acting synergistically in some cases. Sig- nificant extracellular metallothionein was observed when copper or zinc and copper were present in the culture medium, most of which is likely a consequence of cell leakage because no evidence of physiological secretion was observed. Total metallothionein pro- duction (cellular and extracellular metallothionein levels) indicated that copper was a better metal- lothionein inducer than zinc in these experimental conditions. These results indicate that metallo- thionein regulation in the hepatocyte is different depending on the extracellular metal levels and com- position and that attention must be given to metal- lothionein release from the hepatocyte. (HEPATOLOGY 1991;14:648-654.)

The liver is believed to be a central organ in zinc (Zn) and copper (Cu) homeostasis in the organism (1, 2). To characterize how the liver regulates the metabolism of these essential heavy metals, the use of cultured hepatocytes has proved to be a useful tool. Several comprehensive studies on the hepatocyte metabolism of Zn and Cu have been published (3-11). In these studies metallothionein (MT), a low molecular weight heavy metal-binding protein, was usually considered to play a central role. However, its levels were either not mea- sured or measured by rather insensitive or nonspecific

Received June 1, 1990; accepted May 22, 1991. Supported by National Institutes of Health grant ES 01629. Juan Hidalgo also acknowledges the support of the Fondo de Investigaciones

Sanitarias de la Seguridad Social (90/0065-2-D). Address reprint requests to: Juan Hidalgo, Ph.D., Departamento de Biologia

Celular y Fisiologia, Facultad de Ciencias, Universidad Aut6noma de Barcelona, Bellatema, Barcelona 08193, Spain. 3 1/1/3 169 1

techniques; moreover, uncertainty even exists about whether Cu induces de nouo synthesis of MT in cultured hepatocytes (2).

The putative secretion of MT by cultured hepatocytes has never been studied in detail. This is noteworthy because experimental studies suggest a normal presence of circulating MT (12-21), the source of which, at least in part, might be the liver (22). Biliary excretion of MT has also been reported (16, 23). Studies on the putative secretion of MT by hepatocytes therefore are needed to establish such a possibility.

Therefore the aim of this work was to study the effect of physiological (10 p,moliL) and nonphysiological (0, 1, 50 and 100 FmoVL) concentrations of Zn and/or Cu in the culture medium (in the presence of the physiological chelator albumin) on hepatocyte MT content and the putative MT secretion in those conditions (the terms physiological and nonphysiological for metal concentra- tions are relative; see Discussion).

MATERIALS AND METHODS Isolation and Maintenance of Hepatocytes. Isolated liver

parenchymal cells were prepared from adult male Lou rats by using the three-step perfusion technique of Garvey and Heil (24) as modified from Seglen (25) and Knook, Sleyster and van Noord (26). The cells were isolated between 11 AM and 1 PM and had greater than 85% viability as judged by trypan blue dye exclusion. The hepatocytes were washed several times in Leibovitz L-15 medium (27) containing 20 mmol/L HEPES, streptomycin (100 pg/ml), penicillin (60 pg/ml) and gen- tamycin (50 pg/ml) to remove nonparenchymal cells. Paren- chymal cells were finally suspended (1 x lo6 cells/ml) in L-15 medium supplemented with 20 mmol/L HEPES, 5 mmol/L NaHCO,, streptomycin (100 pg/ml), penicillin (60 pg/ml), gentamycin (50 pg/ml), insulin (1 pg/ml) and 10% FCS. Four milliliter aliquots of this suspension were pipetted onto 60-mm plastic culture dishes that had previously been coated with collagen (Vitrogen-100, Collagen Corp., Palo Alto, CA), and viable parenchymal cells were allowed to attach selectively for a 2-hr incubation period at 37" C in a 5% CO, atmosphere. The medium was then removed and the cells washed once with 3 ml of L-15 wash medium. Three milliliters of fresh L-15 medium, supplemented with 20 mmol/L HEPES, 5 mmol/L NaHCO,, streptomycin (100 pg/ml), penicillin (60 pg/ml), gentamycin (50 pg/ml), BSA (2 mg/ml) and 10 pmol/L ZnSO, to approx- imate a physiological plasma Zn concentration, were added to the culture dishes. The hepatocytes were then incubated for about 20 hr at 37" C in a 5% CO,, H,O-saturated atmosphere.

648

Vol 14. No 4. Pt 1. 1991 KOI,E OF ZN AND Cc' ON IIEPRTOCYTE MT REGULATION 649

Role of Zn and Cu on Hepatoryte M T Regulation. After the equilibration period (approximately 20 hr) , the hepatocytes were incubated with the BSA-containing L-15 medium alone or supplemented with Zn (1, 10.50 and 100 FmoliL), Cu ( 1 , l O and 50 pmol/L) or Zn + Cu ( I . 10. and 50 FmoVL of each metal) for 24, 48 or 72 hr. The media were changed daily. The medium from each culture dish was collected every 24 hr and centrifuged (3,000 g, 10 min) at 4" C to eliminate detached cells; the supernatant was stored at - 20" C until MT assay. After completion of the experimental periods, the cells were routinely washed twice with 3-ml aliquots of an EDTA buffer (10 m m o w HEPES, 10 mmoliL EDTA, 150 mmol/L NaCl, pH 7.4) followed by two 3-ml aliquots of cell wash buffer (10 mmol/L HEPES, 140 mmoliL NaC1, 7 mmoVL KCl, 1 mgiml glucose, pH 7.4), as previously described (9) . The EDTA treatment does not interfere with the MT determination because the RIA used is able to measure both the apo- and the metallo-form of the protein (see below). Two milliliters of PBS were then added and the cells scraped with a rubber policeman. The hepatocytes were stored at ~ 20" C until MT assay. MT measurements were normalized to a per milligram cell protein basis and are shown as amounts accumulated24 hr. Each experiment was repeated with two separate hepatocyte prep- arations, and the results were analyzed with the pooled data. Usually the data are expressed as means t S.E.M. of 5 to 18 determinations, except for lactate dehydrogenase (LDH ), where n = two to three.

Assays. After thawing, the hepatocytes were sonicated for 20 sec at 4" C and centrifuged (3,000 g, 10 min) at the same temperature; the supernatant was stored at ~ 20" C. Cellular and extracellular MT were analyzed by a highly specific and sensitive RIA previously described ( 14,28-30 ). LDH content of the medium (units per liter per microgram cell protein) was measured as described by Bergmeyer and Bernt (31); neither Zn nor Cu (alone or together) affected LDH measurements at the metal concentrations studied (up to 100 pmoliL). The metal concentration of the media was confirmed by flame atomic absorption spectrophotometry. Cellular protein content was determined as described by Bradford (32). Results were analyzed by one-way or two-way ANOVA or with the Kruskal-Wallis one-way ANOVA, using the pooled data of the two hepatocyte preparations performed.

Materiah. Cell culture media and FCS were obtained from Gibco (Grand Island, NY). All chemicals were reagent grade. Glucagon-free insulin was a gift from Dr. William W. Bromer. Lilly Research Laboratory, Indianapolis. IN.

RESULTS The control and experimental groups of cells exhibited

no differences in morphological characteristics (i.e., all had the flattened, polygonal appearance assumed by hepatocytes on attachment to a collagen matrix). After 20 hr of culture, the mean cytosolic protein levels of the culture dishes were 527 i 78 pg ( n = 2) and 287 ? 24 p.g (n = 3) for the first and second hepatocyte prepara- tions, respectively, a figure that agrees well with the value of 1.2 to 1.3 mg for total protein previously reported (6). Cell attachment levels were clearly affected by the time of culture, and the metal treatments were studied (Fig. 1). Two-way ANOVA with time of culture and metal concentration as main factors indicated that the percentage of cells attached (as measured by the cytosolic protein present in the dishes) decreased signif- icantly regardless of the metal treatment (p < 0.001);

1 2 3

:$ \ + t 't

I 1 2 3 1 2 3

I Days

FIG. 1. Percentage of cytosolic protein remaining in the culture dishes of hepatocytes incubated under various conditions. Hepatocytes were incubated in L-15 medium containing 2 mgiml BSA and 10 FmoliL Zn2 ' for up to 20 hr; the cytosolic protein present after this incubation is considered 100% (day 0 ) . The medium was then changed and new BSA-containing, L-15 medium supplemented with no metal 1 0 ) . or with Zn (:.), Cu (,A ) or Zn -1- Cu (m) was added. The metal concentrations studied were 1, 10 and 50 FmoliL. When Zn + Cu were used, the concentrations stated are of each metal. The hepatocytes were incubated under those conditions for up to 3 days; the medium was changed daily. The percentage of cytosolic protein remaining in the dishes compared with day "0" is shown. Each point represents mean 2 S.E.M. In = 5 to 6i from two separate hepatocyte prepara- tions. For statistical significances see "Results."

the presence of Zn in the culture medium significantly increased (p < 0.002) the percentage of cells attached to the dishes, whereas the presence of Cu decreased it (p < 0.001). The interaction between both factors was significant (p < 0.002) for the latter. Those dishes incubated in the presence of Zn + Cu showed interme- diate levels of cell attachment because Zn partially reversed the effect of Cu after 2 (p < 0.001) and 3 ( p < 0.025) days of culture. We do not know why cells detach from the plates, but this could be related to the integrity of the membranes, as the effect of Zn (de- creasing) and Cu (increasing) on cell detachment levels suggests. The former is known to stabilize the mem- branes (33-35), whereas the latter has the opposite effect (34). Whether other divalent metals have an effect on hepatocyte detachment remains to be established. On the other hand, it is noteworthy that cell detachment could not be directly related to cell death in all cases, at least in these experimental conditions, as revealed by the LDH results (see below).

Figure 2 shows the LDH content of the media of the hepatocytes incubated with no metals or with 50 pmoVL metal (Zn, Cu or Zn t Cu). Whereas Zn significantly (p < 0.001) decreased LDH levels, Cu increased them (p < 0.001). Those hepatocytes incubated with Zn + Cu also showed increased extracellular LDH levels (p < 0.001), but they were lower than those shown by

650 HIDALGO, DINGMAN AND GARVEY HEPATOLOGY

L DH I

0.5

A

1 2 3 Days

FIG. 2. LDH content (units/liter/microgram cell protein) of the media of the hepatocytes incubated with no metals or with 50 pmol/L metal. The symbols are the same as those of Figure 1. LDH levels of the hepatocytes after the period of recovery are also shown (0). Each point represents mean r S.E.M. (n = 2 to 3) of one hepatocyte preparation. For statistical significances see “Results.” = no metal; A = Zn; A = Cu; 8 = Zn i Cu.

Cellular MT

Days

FIG. 3. MT content of hepatocytes incubated under various conditions. Hepatocytes were incubated in L-15 medium containing 2 mg/ml BSA and 10 KrnoUL Zn2 + for up to 20 hr (0). The medium was then changed and new BSA-containing, L-15 medium supplemented with no metal (0) or with Zn ( A ) , Cu (A) or Zn + Cu (8) was added. The metal concentrations studied were 1,lO and 50 Kmol/L; when Zn + Cu were used, the concentrations stated are of each metal. The hepato- cytes were incubated under those conditions for up to 3 days; the medium was changed daily. Each point represents mean 2 S.E.M. (n = 2 to 3) from one of two separate hepatocyte preparations. MT levels are amounts accumulated/24 hr. For statistical significances of the pooled data from the two hepatocyte preparations see “Results.”

0 50 100

IJM

FIG. 4. MT induction by Zn, Cu or Zn + Cu in hepatocytes incubated for 1 day with increasing metal concentrations. Hepatocytes were incubated in L-15 medium containing 2 mg/ml BSA and 10 pmol/L Zn” for up to 20 hr. The medium was then changed and new BSA-containing, L-15 medium supplemented with no metal or with Zn ( A ) Cu ( A ) or Zn + Cu (8) was added. Twenty four hours later the cellular MT content was measured; MT levels are shown as a percentage of those shown by hepatocytes incubated without extra- cellular metals. Each point represents mean ? S.E.M. (n = 5 to 6) from two separate hepatocyte preparations. For statistical signifi- cances see “Results.”

hepatocytes incubated with Cu alone (p < 0.001), sug- gesting that Zn partially reversed the effect of Cu. This is in agreement with the proposed effects of Zn and Cu on the integrity of the membranes (see above). The combined results of cell detachment and LDH levels suggest that the former is not always directly related to cell death. For example, cell detachment increased with time of culture in control (cultured without metals) and Zn-treated hepatocytes (to a lower extent), but LDH levels in the media did not increase; actually, they decreased. If detached cells were dead, a huge amount of LDH should be present in the culture medium, which was not the case in those culture conditions. In contrast, Cu clearly was damaging the cells, as revealed by increasing amounts of LDH in the medium in addition to increased cell detachment levels.

Figures 3 and 4 show the hepatocyte content of MT of all the experimental groups. Two-way ANOVA with time of culture and metal concentration as main factors indicated that the hepatocyte MT content increased steadily with time of culture regardless of the metal treatment (p < 0.001) and that the presence of Zn, Cu or Zn + Cu in the culture medium increased hepatocyte MT levels significantly (p c 0.001) in general (Fig. 3). Zn and Cu alone increased the hepatocyte MT content, in a limited dose-response manner, to a similar extent (Fig. 4). The percentage of hepatocyte MT compared with the hepatocytes cultured without metals was approximately the same in all the time periods studied (up to 3 days, not shown). In contrast, the presence of both metals in the medium had a clearer dose-response effect on cellular MT levels; these were significantly (p < 0.001) higher than those measured after incu-

Vol 14, No 4, Pt 1 1991 KOI,E OF ZN AND CI ON HEPATOCYTE MT REGULATION 65 1

Extracellular MT

I 50 lJM 5

1 2 3 1 2 3 1 2 3 Days

FIG. 5. Extracellular M‘T content of the hepatocytes described in Figure 3. The medium from each culture dish was collected every 24 hr and centrifuged a t 4” C to eliminate detached cells; the supernatant was used for MT assay. MT levels are amounts accumulatedi24 hr. Each point represents mean k S.E.M. In = 2 to 9, from one of two separate hepatocyte preparations For statistical significances of the pooled data from the two hepatocyte preparations see “Results.” The symbols are the same as those of Figure 3

bation of hepatocytes with Zn or Cu alone. In some cases (see Fig. 3) a synergistic effect was evident. (i.e., at 10 and 50 pmol/L, second and third day of culture).

Figure 5 shows the MT present in the media of all the experimental groups normalized to a per milligram cytosolic protein basis for the culture in which MT was measured. When this was not possible (i.e., the 24 and 48 hr levels of MT in the media of the hepatocytes to be harvested at 72 hr), the protein content used to normalize extracellular MT was the mean content of the plates harvested in those time periods. Two-way ANOVA with time of culture and metal concentration as main factors revealed that extracellular MT content increased steadily with time of culture regardless of the metal treatment (p < 0.001). Whereas the presence of Zn in the medium consistently decreased the extracel- Mar MT levels, that of Cu increased them (p < 0.001 in both cases). The presence of both Zn and Cu in the medium further increased extracellular MT levels (p < 0.001). Figure 6 shows extracellular MT levels. normalized to a per-LDH content basis, of the hepato- cytes incubated with no metals or with 50 pmol/L metal (Zn, Cu or Zn + Cu). Expressed in this way, extracel- lular MT content was quite proportional to the cellular MT content. Both Cu and Zn increased extracellular MT levels (p a t least < 0.025). At this metal concentration. Cu increased extracellular M T levels to a higher extent than Zn (p < 0.001) (the same was true for cellular MT, although considering all the metal concentrations no statistical significance is achieved; see above). The presence of Zn + Cu in the medium further increased extracellular MT levels ( p < 0.001).

Figure 7 shows the total MT production (cel- lular + extracellular MT) of the hepatocytes of all the experimental groups. The results were analyzed as before; they were quite similar to those of cellular MT

1 2 3

Days

FIG. 6. Extracellular MT content, normalized to a per LDH content basis, of the hepatocytes incubated with no metals or with 50 pnoliL metal. The medium from each culture dish was collected every 24 hr and centrifuged at 4“ C to eliminate detached cells; the supernatant was used for MT and LDH assays. MT levels are amounts accumu- latedi24 hr. The symbols are the same as those in Figure 3. MT levels of the hepatocytes after the period of recovery are also shown. Each point represents mean ? S.E.M. In = 2 to 3) of one hepatocyte preparation. For statistical significances see “Results.”

.E 1.6 0, 4-8

2 n

= 1.2 Q,

0 I

0 C

1 0.8

0.4

1 2 3

J , I . *

1 2 3

50 p~

Days

FIG. ‘i Total MT production (cellular plus extracellular MT) of hepatocytes incubated with no metals, Zn. Cu or Zn + Cu for up to 3 days. MT levels are amounts accumulatedi24 hr. Each point represents mean c S.E.M. ( n = 2 to 3) from one of two separate hepatocyte preparations. The symbols are the same as those of Figure 3. For statistical significances of the pooled data from the two hepatocyte preparations see “Results.”

levels (Fig. 31, except that extracellular Cu consistently increased total MT production compared with that shown by hepatocytes incubated with Zn, suggesting that the former is a better MT inducer in the present

652 HIDALGO, DINGMAN AND GARVEY HEPATOLOGY

experimental conditions. The presence of both Zn and Cu further increased total MT production, also in a dose-dependent manner.

DISCUSSION Cellular MT. Both Zn and Cu are usually present in

the plasma in concentrations of approximately 10 pmoVL; most of the metals, however, are not in free form but bound to plasma proteins. Thus, about two thirds of the Zn and 5% of the Cu is bound to albumin, whereas 90% of the Cu is bound to ceruloplasmin (1). However, a substantially higher percentage of Cu should be bound to albumin in the portal blood because it appears that this protein is responsible for the initial transport of Cu to the liver after absorption by the intestine (36). The same was previously demonstrated for Zn (37, 38). This suggests that a more physiological approach to the role of Zn and Cu on hepatocyte MT regulation occurs when the metal is presented to the hepatocyte bound to the physiological chelator(s1. In this study, we used albumin because it binds both Zn and Cu in physiological conditions and because it has been the chelator used by most of the previous literature on metal uptake by hepatocytes (3, 5, 9-11, 36, 39). The dose of albumin used, 2 mg/ml (30 p,moVL), is about 10 times lower than the physiological albumin concentration (40). However, the buffering capacity of BSA relative to the total (exchangeable) metal ion is maximal and therefore it is expected that most of the Zn will be bound to BSA (40). Our results indirectly support this assumption (see below). Being conservative, however, it might be said that albumin will be acting as a chelator at least for the 1 and 10 p,mol/L concentrations of Zn and Cu (alone or together). Keeping in mind the above statements, 10 p,moVL concentrations of Zn, Cu or Zn + Cu (each) have been called above physiological, although in the case of Cu, the 1 p,mol/L concentration might be closer to the actual physiological situation.

Hepatocytes maintained with no readily detectable metal levels in the medium (as determined by flame atomic absorption spectrophotometry) showed a steady increase of cellular MT levels throughout the time period studied. This effect may be related to the progressive transformation of the hepatocyte that occurs with culture time, but no supporting data exist. What is clear is that MT levels were not decreased by culture conditions of Zn deficiency (although the question remains whether nondividing hepatocytes would arrive at Zn deficiency in the time periods studied). This is in contrast to conclusions derived from in uiuo studies (1). Although trace levels of Zn in the medium could maintain, in some degree, the normal MT metabolism in the hepatocyte, it seems that this metal has in fact little importance on MT regulation in cultured hepatocytes in the presence of the physiological chelator BSA. As measured by RIA, MT content of hepatocytes maintained in media supplemented with 1, 10,50 or 100 p,mol/L Zn was only slightly greater (up to twofold) than that shown by hepatocytes maintained without Zn in the medium at all times studied. That is,

hepatocyte MT content showed little dependence on the extracellular Zn content, even when this was much higher than a physiological plasma Zn concentration (approximately 10 p,mol/L). This is in disagreement with previous reports (41, 42), which indicated a higher MT induction in hepatocytes cultured with Zn concentra- tions similar to those reported here. However, some of these studies were made without albumin present in the medium (41, 42). Absence of albumin in the medium could cause an unphysiological Zn accumulation in the hepatocyte, which in turn would increase MT levels. This hypothesis is supported by the results of Pattison and Cousins (91, who have demonstrated that in the presence of 2 mg/ml of BSA total hepatocyte Zn levels show a small dependence on medium Zn concentration (2 to 71 p,mol/L) during a 20-hr incubation period, suggesting that the hepatocyte is capable of controlling and maintaining specific intracellular Zn levels. Re- cently, the same laboratory (10) has demonstrated a much greater Zn accumulation in hepatocytes incubated for 18-hr with medium containing varying concentra- tions of Zn (1 to 48 p,mol/L). In this case, however, the experiments were made immediately after cell isolation and attachment, that is, without an equilibration period. MT levels, measured by the Cd (11) binding method, showed a 7.5-fold increase at 48 p,mol/L Zn (10). This MT increase is similar to that shown by hepatocytes incubated with medium without BSA (41,42). This is in contrast to the twofold increase at 50 p,mol/L Zn of the present report. Collectively, all these data suggest that hepatocyte MT is only strongly induced by elevated Zn concentrations in the culture medium when the hepa- tocyte cannot adequately regulate the intracellular Zn levels. Such net accumulation of Zn presumably occurs in the absence of the physiological chelator albumin or when the cells are not allowed to recover from the isolation procedure itself.

Hepatic MT induction by Cu in uiuo has clearly been demonstrated, but very little attention has been given to the role of Cu on MT regulation in cultured hepatocytes, with even uncertainty about the actual de nouo synthesis of MT in this system (2). The MT content of hepatocytes maintained in media supplemented with 1, 10 or 50 p,mol/L Cu, as measured by RIA, was slightly but significantly greater than that shown by hepatocytes maintained without metals in the medium at all times studied. MT levels were in general similar to those shown by hepatocytes maintained with Zn; as for Zn (91, Cu accumulation in the hepatocyte appears to equili- brate by exchange with Cu pools (5), a process where albumin may play an important role (1,8,11,36,39,43). Collectively, these data suggest that in physiological conditions the hepatocyte is equally capable of con- trolling and maintaining specific intracellular Cu levels, as it does with Zn levels. This could also explain, as with Zn, the small dependence that the hepatocyte MT shows on the Cu concentration of the culture media. However, substantial differences between the effect of Zn and Cu are seen when MT release by the hepatocytes is considered (see below).

Vol. 14, No 4. l’t 1, 1991 KOLE OF ZN AND C U ON HEPATOCYTE MT REGULATION 653

To our knowledge, the combined effect of Zn and Cu on hepatocyte MT content has never been tested. This is surprising because both metals are usually present in the plasma in similar concentrations (approximately 10 pmolb). In contrast to Zn or Cu alone the supplemen- tation of the culture media with both metals ( 1 , l O or 50 pmol/L of each metal) had an important effect on the hepatocyte MT content, especially after 48 and 72 hr of incubation. MT levels were consistently increased with all metal concentrations studied. The MT increase was higher as the metal concentration in the medium increased (i.e., a synergistic effect between Zn and Cu was clearly seen at 10 and 50 p,mol/L). These results are unlikely to be due to a higher accumulation of any of the two metals because it has clearly been demonstrated that incubation under conditions of 0 to 20 pmolL. Zn in the medium had no influence on Cu accumulation by hepatocytes and vice versa (5). However, this has been demonstrated for incubations of hepatocytes for approx- imately 20 hr. Because in our study the greatest increase of MT levels was observed after 48 and 72 hr of incubation, a participation of metal accumulation on MT induction during those periods cannot be ruled out. Alternately, it is important to keep in mind the results obtained with Zn-deficient rats (44, 45 1, which demon- strated that the induction of liver MT by Cu was identical in Zn-supplemented and Zn-deficient rats and that the half-life of the Cu-MT was lower (12 hr) in the latter than in the former rats (17 hr ) , suggesting a decreased stability of the Cu-MT in the presence of reduced Zn levels. This could account for the synergistic effect of Zn + Cu on hepatocyte MT levels. In any case, these results suggest that MT regulation in the hepato- cytes appears to differ when both metals are present simultaneously in the culture media. Therefore a more physiological approach indicates that both Zn and Cu should be present in the culture media when studying MT regulation by agents such as the endocrine system.

Extracellular MT. Evidence from animal studies, as previously mentioned, indicates a normal presence of circulating MT, the source of which might be, at least in part, the liver; biliary excretion of MT has also been demonstrated. However, the putative MT secretion by hepatocytes maintained in culture has never been studied in detail (46). which aimed us to measure extracellular MT levels under the present experimental conditions.

A strong presence of MT was detected in the medium. but in general it paralleled cellular MT levels. This was particularly evident when extracellular MT levels were normalized to a per LDH content basis because then they were quite proportional to the cellular MT levels, suggesting that extracellular MT mostly reflects cell leakage. Other experimental approaches, such as using Primaria flasks instead of collagen-coated plates or the addition of hormones (corticosterone, dexamethasone, adrenaline or insulin alone or combined, one or several additions, always in the presence of 30 pmol/L albumin and 10 pmol/L Zn) failed to give significant evidence of physiological MT secretion by cultured hepatocytes

(results not shown). We must conclude that no evidence exists, a t least in our study, that hepatocytes in primary cultures secrete MT. This is in agreement with the site of MT synthesis, free polyribosomes, which is not consistent with a secreted protein (47). This raises the question of whether the in vivo situations where serum or plasma MT levels are increased are damaging the liver, a possibility previously suggested (15). However, small amounts of MT may actually be secreted by cultured hepatocytes and not be measured because of the presence of large amounts of MT leakage in the present experimental conditions, which deserves further attention. Alternately, the source of serum MT might not be the liver but other tissues.

Total MT Production. The main conclusion of our study is that the cellular integrity and the release of MT to the culture medium must be taken into account in any future work on MT. Otherwise we could be led to serious misinterpretation of the results. (i.e., if one takes into account only the cellular content of MT, Cu appears to be as weak an inducer of MT as Zn; in contrast, taking into account both extracellular and intracellular MT, it is clear that Cu is a better MT inducer in the hepatocyte than Zn). The combined presence of both metals causes a greater increase of the MT production, in some cases ii.e., after 2 or 3 days of culture) showing a synergistic effect; this might be due to the higher or better metabolic capacity of the hepatocyte, given the importance of Zn in normal cell function or, alternately, to the stabilization of the MT protein, as in vivo studies suggest (44,45). It is also noteworthy that the production of MT in the hepatocyte is increased as the culture time increases, with independence of the extracellular metal content and composition.

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