(Received for publication, September 20, 1994; and in revised form, January 3, 1995)
From the
Embryonic cells from transgenic mice with targeted disruption of metallothionein I and II genes expressed no detectable metallothionein either constitutively or after treatment with cadmium, in contrast to cultured cells that were wild type or heterozygous for the loss of the metallothionein genes. Metallothionein null cells were most sensitive to the cytotoxic effects of cadmium, the membrane permeant oxidant tert-butylhydroperoxide, and the redox cycling toxin paraquat. No marked differences were seen among the wild type, heterozygous, or metallothionein null cells in glutathione levels or in the activity of CuZn-superoxide dismutase, glutathione peroxidase, or catalase. Nevertheless, metallothionein null cells were more sensitive to tert-butylhydroperoxide-induced oxidation as ascertained by confocal microscopic imaging of dichlorofluoroscein fluorescence. These results indicate basal metallothionein levels can function to regulate intracellular redox status in mammalian cells.
Metallothioneins (MT) ()are ubiquitous low molecular
weight proteins characterized by their unusually high affinity for
metals and rich cysteine content. First identified as cadmium-binding
proteins (Hamer, 1986), MT are now known to form high affinity
complexes with an assortment of trace metals including mercury,
platinum, and silver, as well as biologically essential metals like
zinc and copper (Hamer, 1986). In many cells MT represents the single
most abundant protein thiol source and the major zinc-binding protein.
Basal MT levels can be increased by metals. Treatment of animals or cells with zinc, copper, or cadmium markedly increase MT protein by activation of transcription factors that recognize metal-responsive elements located in the 5` untranslated region of the MT gene. Although there is general agreement that MT can protect organisms against heavy metal toxicity, it is unlikely that this is the only or even the primary function of MT because these ions are not generally present either endogenously or environmentally in high levels and basal levels of MT are found in most cells. Moreover, gratuitous metal and nonmetal inducers, such as cytokines and drugs, have been identified, which do not bind directly to MT. Thus, MT have been postulated to participate in zinc and copper homeostasis, to regulate the synthesis and activity of zinc metalloproteins, most notably zinc-dependent transcription factors, to protect cells against electrophilic anticancer drugs and mutagens, and to guard against reactive oxygen intermediates (Karin, 1985; Basu and Lazo, 1990).
To test these hypotheses, two general approaches have been used: pharmacologic and genetic. When animals or cells have been treated with pharmacologic or toxicologic inducers of MT, somewhat conflicting conclusions have emerged, perhaps because the agents used have multiple targets (Hamer, 1986; Karin, 1985; Basu and Lazo, 1990). Gene transfer methods also have failed to produce a consensus regarding MT functionality, possibly because of complex and poorly understood MT dose effects or because of additional cellular factors necessary to complement MT.
The widespread phylogenic and cellular expression of MT and its extensive putative functions led most investigators to conclude MT is essential for development. Thus, the recent successful production of MT null mice was surprising (Michalska and Choo, 1993; Masters et al., 1994). In initial studies, these mice reproduced normally and displayed few overt abnormalities other than increased sensitivity to cadmium toxicity. We now report on the phenotypic characteristics of embryonic cells isolated from these mice.
Figure 1: MT levels in MEC. MT was measured by radiolabeled cadmium binding in heat-stable low molecular protein extracts from MEC. The mean results of three or more determinations are shown. Bars equal S.E. Hatched symbols are MT +/-, and blacksymbols are MT +/+. n.d. = not detectable.
The morphology of the three MEC were similar when the cells were cultured with 20% fetal bovine serum in complete medium (Fig. 2). A 24-h exposure to 30 µM cadmium was associated with considerable cytotoxicity in MT -/- but not MT +/+ cells; MT +/- cells had an intermediate response to cadmium (Fig. 2). Deletion of MT I and MT II genes did not significantly alter levels of the major nonprotein thiol GSH nor the activity of the antioxidant enzymes CuZn-superoxide dismutase, glutathione peroxidase, or catalase in total cell homogenates (Table 1). To exclude potential interference in glutathione peroxidase assay from endogenous MT in wild type and heterozygous cells, we also examined high molecular mass homogenates (>10 kDa) and found no significant differences in the glutathione peroxidase activities (data not shown).
Figure 2:
Morphology of wild type, heterozygous and
MT null cells after treatment with cadmium. MT -/-,
+/-, and +/+ cells were treated with vehicle or 30
µM CdCl for 24 h. Bar = 50
µm.
Figure 3:
Survival of MEC after CdCl,
tBH, or paraquat treatment. Cells were incubated with CdCl
(panelA), tBH (panelB), or
paraquat (panel C) for 1 h, and cell survival was measured
4-6 days later by a colorimetric assay. The symbols represent the
mean values from 8 (panels A and B) to 16 (panel
C) determinations. Bars = S.E., unless smaller
than the symbol. Opencircles, -/-; opensquares, +/-; closedcircles +/+.
The sensitivity of null cells to tBH was examined further using the oxidant-responsive fluorescent probe DCFHDA. Cells were preloaded with 10 µM DCFHDA for 15 min and then treated briefly with 500 µM tBH. The conversion of DCFH to DCF was monitored by the appearance of a fluorescent signal in live cells using confocal microscopy and provided relative quantitative information regarding the steady-state oxidant burden. The basal level of DCF formation in MT -/- cells was not significantly greater than in either heterozygous or wild type cells. Within 1 min after tBH, additional marked intracellular oxidation of DCFH occurred ( Fig. 4and 5). In MT null cells, a 10-fold increase in fluorescence over the initial fluorescence was sustained for at least 8 min after tBH treatment (Fig. 5). A 8- and 5-fold increase, in heterozygous and wild type MEC, respectively, was observed during the tBH treatment (Fig. 5). At all times the most intense fluorescent signal in MT -/- cells was localized centrally in an area corresponding to the nucleus determined by simultaneous phase microscopy (Fig. 4).
Figure 4: Formation of dichlorofluoroscein in MEC. Cells were exposed to 500 µM tBH and the intracellular fluorescence determined 480 s later microscopically. A, MT +/+ untreated; B, MT -/- untreated; C, MT +/+ treated with 500 µM tBH; D, MT -/- treated with 500 µM tBH. Bar = 30 µm.
Figure 5: Kinetics of dichlorofluoroscein formation in MEC. Representative results from four independent experiments were normalized to initial fluorescent values. The mean fluorescence values obtained from 15-20 cells are shown with S.E. tBH was added after the second determination at 60 s. Circles, MT -/-; triangles, MT +/-; squares, MT +/+.
The ubiquitous cellular and phylogenic distribution of MT implies important physiological functions. These putative functions remain unclear in spite of a wealth of structural and molecular biological information regarding MT. Nonetheless, a role for MT in detoxifying heavy metals is apparent (Hamer, 1986; Karin, 1985), and support for a protective role of MT against reactive oxygen species is accumulating (Thornalley and Vasak, 1985; Sato and Bremner, 1993; Chubatsu and Meneghini, 1993; Tamai et al., 1993). MT is thought to have a detoxifying role against metals such as cadmium because: (a) MT transcription is induced by cadmium (Karin, 1985); (b) MT forms high affinity thiolate clusters with cadmium, which reduces the ability of the metal to react with other biomolecules (Kagi and Schaffer, 1988); (c) cultured cells selected for resistance to cadmium have elevated MT levels (Hamer, 1986; Basu and Lazo, 1990; Kelley et al., 1988); and (d) overexpression of MT after gene transfer reduces the sensitivity of a variety of cells to cadmium (Basu and Lazo, 1990; Kelley et al., 1988; Kaina et al., 1990; Koropatnick and Pearson, 1993). The most compelling support, however, is derived from two independent studies in which targeted disruption of MT I and MT II genes in mice enhances their sensitivity to cadmium (Michalska and Choo, 1993; Masters et al., 1994). In the current report, we demonstrate that embryonic cells isolated from a transgenic mouse deficient in MT I and MT II genes retain enhanced sensitivity to cadmium ( Fig. 2and Fig. 3).
In contrast to the
involvement of MT in heavy metal detoxification, support for a role for
MT as an antioxidant is more circumstantial, ambiguous, and
controversial. Nonetheless, there are several reasons for believing MT
could function as an antioxidant in mammalian cells. First, a variety
of physical and chemical stresses known to be associated with oxidative
injury increase MT gene expression. X-irradiation (Koropatnick et
al., 1989), hyperoxia (Veness-Meehan et al., 1991), and
restraint (Bauman et al., 1991) result in tissue-specific
increases in oxygen free radicals and mRNA for MT expression.
Xenobiotics, such as paraquat (Bauman et al., 1991) and
CCl (Hidalgo et al., 1988), and anticancer drugs,
such as doxorubicin, cisplatin and bleomycin (Basu and Lazo, 1990;
Bauman et al. 1991), have been reported to have similar
effects in vivo. These stimuli, however, can cause an
inflammatory response characterized by the production of cytokines,
such as interleukin-1, interleukin-6, tumor necrosis factor
, and
interferon
, which can increase MT gene expression (Hamer, 1986;
Karin, 1985; Schroeder and Cousins, 1990; De et al., 1990).
Thus it is unresolved if reactive oxygen radicals per se or
secondary cytokines affect MT expression in vivo. It is
intriguing, however, that MT expression has recently been found to be
responsive to oxygen tension in cultured tumor cells (Murphy et
al., 1994). Second, MT induction with heavy metals or cytokines
reduces cell sensitivity to oxidant injury caused by ionizing radiation
(Basu and Lazo, 1990), hydrogen and organic peroxides (Schwarz et
al., 1994; Mello-Filho et al., 1988), hyperoxia (Hart et al., 1990), CCl
(Schroeder and Cousins, 1990),
and tumor necrosis factor (Leyshon-Sorland et al., 1993).
Heavy metals and cytokines are promiscuous transcriptional activators,
however, and cadmium or zinc pretreatment can produce an
H
O
-resistant cell as a result of activating
non-MT thiol antioxidants (Chubatsu et al., 1992). Third,
overexpression of MT via direct gene transfer has been shown to reduce
the sensitivity of lower (Tamai et al., 1993) and higher
(Schwarz et al., 1994) eukaryotes to oxidative injury. Other
investigators, however, have failed to detect altered resistance to
oxidative stress in Chinese hamster ovary cells that overexpress human
MT IIA after gene transfer (Kaina et al., 1990), suggesting
the protection offered by MT to oxidant injury may be complex. Fourth,
underexpression of MT with antisense oligonucleotides restores the
H
O
sensitivity of Chinese hamster lung
fibroblasts with previous elevated nuclear MT content (Chubatsu and
Meneghini, 1993).
MT null mouse cells provide a more direct and simple model with which to examine the role of MT as a member of the intracellular antioxidant network. The ability to totally deplete cells of MT I and MT II protein has not been achieved with antisense techniques (Leibbrandt et al. 1994). Moreover, we were unable to express MT protein even with a strong stimulus such as cadmium (Fig. 1), in contrast to either the heterozygous or wild type cells. Thus, the MT null cells present a useful model to dissect the effects of heavy metals in the absence of MT induction. Finally, the MT -/- cells were similar to the control cells with respect to activity of several important antioxidants (Table 1).
In a previous report (Schwarz et al., 1994), we noted MT overexpression after plasmid transfection reduced the sensitivity of NIH3T3 cells to tBH, a membrane permeant oxidant thought to kill mammalian cells by peroxidizing membrane lipids (Masaki et al., 1989). tBH-mediated DNA damage was not reduced in cells overexpressing MT (Schwarz et al., 1994). This oxidant can produce a complex variety of reactive oxygen intermediates within cells (Royall and Ischiropoulos, 1993; LeBel et al., 1992). In the current report, the MT -/- cells demonstrated an enhanced sensitivity not only to tBH but also the redox cycling herbicide paraquat, which is structurally quite distinct from tBH. Although the magnitude of the protection against tBH and paraquat afforded by the presence of both functional MT alleles in the wild type cells may seem small (approximately 2-3-fold) compared to the MT null cells, it is comparable to the protective effect against tBH when MT is overexpressed 4-fold in NIH3T3 cells (Schwarz et al., 1994) or against paraquat when catalase is overexpressed 100-fold in L cells (Speranza et al., 1993) by gene transfer. Therefore, we believe these changes in sensitivity could have biological significance.
The lack of MT resulted in an impaired ability of the
MT null cells to quench the tBH-induced change in fluorescence of the
oxidant-sensitive dye, DCFH. The relative stability of the fluorescent
signal after tBH addition illustrates the advantage of live cell
confocal microscopic methods to detect DCFH oxidation. We saw little
evidence for cellular loss of oxidized DCFH when cells were attached to
coverslips in contrast to previously described studies with detached
cells (Royall and Ischiropoulos, 1993). Although DCFH is a valuable
probe to identify the generation of intracellular reactive oxygen
species, the precise species responsible for the oxidation of DCFH to
DCF are not known (LeBel et al., 1992). It appears unlikely
that superoxide anion or HO
alone directly
oxidize DCFH; H
O
-Fe
-derived
oxidants may be primarily responsible for oxidation of DCFH at least
based on in vitro results (LeBel et al., 1992). The
mechanism by which MT acts as an antioxidant is unclear, although it
can scavenge phenoxyl radicals in vitro as determined by
electron spin resonance (Schwarz et al., 1994). Previous
reports indicated that partially purified MT is capable of scavenging
hydroxyl and superoxide anions in vitro (Thornalley and Vasak,
1985; Thomas et al., 1986; Hainaut and Milner, 1993). Thus,
the cysteine residues of MT might serve as an expendable target for
reactive oxygen species, but such a mechanism does not explain the
failure to see protection against oxidants after MT overexpression in
some studies (Kaina et al., 1990). Alternatively, the
antioxidant properties of MT may rely on its metal speciation and the
ability of oxygen free radicals to release zinc from MT thiolate
clusters and the antioxidant activity of zinc on plasma membranes and
on other nuclear and cytoplasmic proteins (Thomas et al.,
1986; Hainaut and Milner, 1993). It is particularly interesting that
the DCF signal appeared to be localized in a discrete area
intracellularly, most probably corresponding to the nucleus, and that
MT colocalized to this area in the heterozygous and wild type cells.
Clearly more attention should be directed to the functional
significance of nuclear MT.
MT is expressed constitutively at low levels in virtually all cell types. The MT null cells provide a convenient tool to explore various roles of MT without elevating MT levels, either pharmacologically or genetically, to levels that often exceed normal physiological values. Our results indicated loss of MT yielded cells that were more sensitive to tBH and paraquat. Since other antioxidant defense mechanisms appeared not to compensate in the MT null cells, a singularly important value for constitutive levels of MT is apparent. The antioxidant role of MT could suggest the MT null mice may have altered sensitivity to pro-oxidant and inflammatory pathophysiologic states.