The effects of tungstophosphate and tungstosilicate on various stress promoters transformed in Escherichia coli

Journal of Inorganic Biochemistry 94 (2003) 155–160 www.elsevier.com / locate / jinorgbio

The effects of tungstophosphate and tungstosilicate on various stress promoters transformed in Escherichia coli Yutaka Tajima* Clinical Laboratory, Saga Medical School Hospital, Nabeshima, Saga 849 -8501, Japan Received 3 June 2002; received in revised form 14 September 2002; accepted 4 October 2002

Abstract Although tungsten is an important material in some industrial and chemical processes, the biological and biochemical effects, including the toxicity, of tungsten compounds are not known well. In this study, a reporter gene assay using special strains of Escherichia coli was performed to investigate the mode of action of two polyoxotungstates, i.e. undecatungstophosphate (PW11 ) and undecatungstosilicate (SiW11 ). When the bacterial cells were cultured with PW11 , osmY (a stress promoter gene sensitive to osmotic signals) was induced to some extent, while other stress promoters were expressed only slightly. SiW11 gave similar results, but clpB (an analogue of human heat shock protein) was more strongly induced. It is possible that PW11 and SiW11 can produce an osmotic signal at lower concentrations without increasing ionic strength. Since the constituents of PW11 / SiW11 (i.e. HPO 422 , SiO 322 , WO 422 ) showed almost no effect, a chemical feature unique to PW11 / SiW11 and originating from neither of their constituents, i.e. a polyanionic characteristic, may play an important role in their biological effects.  2002 Elsevier Science Inc. All rights reserved. Keywords: Undecatungstophosphate; Undecatungstosilicate; Reporter gene assay; Signal; Osmotic

1. Introduction Although tungsten (W) is a very important material in industry and chemistry, its biological effects, including toxicity, are quite unknown [1–3]. While some prokaryotes are known to require tungsten as a cofactor of several enzymes, it is not thought that tungsten is essential for eukaryotes [4]. Tungsten compounds are used to only a limited extent in biochemistry [1–3]; the details are summarized in the author’s recent review article [5]. The most stable oxidation state of tungsten is W(VI), but only oxoanionic molecular species (such as WO 22 4 ) are stable under aqueous conditions [1,2]. The most familiar tungsten compound is sodium tungstate (Na 2 WO 4 ) and its derivatives. Interestingly, WO 422 has the unique feature that this oxoanion polymerizes easily with itself or with other oxoanions such as phosphate (PO 32 4 ), forming a polyacid called ‘polyoxotungstate’: for example, heating a mixture of WO 22 and PO 32 under acidic conditions causes the 4 4 formation of dodecatungstophosphate ([PW12 O 40 ] 32 , PW12 ) [1,6–9].

PW12 is partially hydrolyzed at neutral pH, and the major polyanion present in the solution is undecatungstophosphate ([PW11 O 39 ] 72 , PW11 ) [6–9]. Although PW11 is decomposed completely at pH.8 [6], 80–90% of this compound still remains as [PW11 O 39 ] 72 below pH 7.2 [10,11]. It therefore seemed natural to investigate the biological and biochemical effects of WO 22 and PW11 , 4 both of which are representative of the tungsten compounds present around physiological (neutral) pH. In the literature, there is some evidence that polyoxotungstates bind with many substances (e.g. proteins, alkaloids) [12–17] and alter their functions and properties [18–20]. Therefore, sometimes such a tungsten compound does have several unique biochemical and / or biological effects even though it is only an inorganic compound. In this study, a reporter gene assay using special strains of Escherichia coli was thus performed to investigate the mode of action of tungsten compounds. 2. Experimental

2.1. Materials *Corresponding author. Fax: 181-952-34-2028. E-mail address: [email protected] (Y. Tajima).

E. coli strains, each of which contained a special stress

0162-0134 / 02 / $ – see front matter  2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0162-0134(02)00595-0

Y. Tajima / Journal of Inorganic Biochemistry 94 (2003) 155–160

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promoter fused to a promoterless structural gene of galactosidase (lacZ), were obtained from Xenometrix (Boulder, USA) as an assay kit known as ‘Pro-Tox’. Culture media were from Difco Laboratory (MI, USA). All other chemicals were obtained from Wako Pure Chemicals (Osaka, Japan).

2.2. Preparation of PW11 /SiW11 PW11 was prepared by partial hydrolysis of PW12 [21,22]. Briefly, H 3 [PW12 O 40 ]?30H 2 O (34 g) was dissolved in 2 M CH 3 COOH (100 ml), and this mixture (heated to 95 8C with vigorous stirring) was neutralized to pH 5.3 by gradual addition of solid KHCO 3 (|20 g). PW11 was purified from this neutralized solution by recrystallization, and dissolved in water to 20 mM and kept at 220 8C until use. Undecasilicotungstate ([SiW11 O 39 ] 82 , SiW11 ) was prepared in a similar way; the details including analytical data of PW11 / SiW11 are described in the author’s reports [22,23].

subculture of each strain (in the same culture medium) was mixed with various chemicals such as PW11 and further incubated (90 min) with vigorous shaking for aeration. After the bacterial cells were lysed with chloroform, bgalactosidase activity of the lysate was measured using o-nitrophenyl-b-D-galactoside as a substrate. Finally, the enzyme activity was corrected by the cell density of each medium (estimated from the optical density at 600 nm), and the extent of the lacZ-fused promoter gene expression was presented as a ratio of the control activity. Orser et al. reported that .30% increase from the control activity can be considered as a significant response [24]. The direct effects of various compounds tested on the b-galactosidase activity were not significant. The assay methods of several serum enzymes and nucleic acids-related enzymes are described in our previous report [25].

3. Results and discussion

2.3. Reporter gene assay and measurement of various enzyme activities

3.1. Effects of PW11 on various stress promoters

The detailed assay procedure is described in the report of Orser et al. [24]. Briefly, E. coli strains, each of which contained a special lacZ-fused stress promoter, were cultured in Luria-Bertani broth (containing 0.2% glucose and 20 mM HEPES buffer, pH 7.0) for 2 h. Then,

The results are shown in Table 1. Effective concentration of polyoxotungstate such as PW11 sufficient to produce a stress response in the E. coli strains was relatively high (|500 mM). This is probably because E. coli has an outer membrane outside the cell wall, which

Table 1 Effects of tungsten compounds on various lacZ-fused promoter gene expression Promoter b

None

Fold induction c (%) Na 2 WO 4 / Na 2 SiO 3 a

PW11 / SiW11

0.5 mM

1 mM

2 mM

5 mM

50 mM

100 mM

200 mM

500 mM

Group 1 (slow) ada d clpB d,e dinB dinD d merR nfo soi28 d umuDC d

100 100 100 100 100 100 100 100

97 / 98 109 / 110 97 / 113 102 / 96 101 / 103 99 / 105 100 / 94 98 / 106

95 / 97 106 / 116 98 / 108 101 / 98 93 / 99 100 / 88 102 / 104 104 / 112

96 / 102 119 / 115 109 / 115 96 / 106 97 / 102 97 / 109 114 / 108 109 / 110

98 / 108 113 / 124 114 / 118 103 / 109 96 / 104 101 / 118 116 / 119 108 / 118

94 / 103 100 / 121 93 / 101 97 / 104 106 / 101 98 / 96 114 / 111 112 / 101

98 / 104 116 / 136 105 / 107 105 / 106 99 / 94 103 / 101 118 / 101 102 / 104

96 / 115 122 / 147 110 / 108 107 / 110 97 / 99 99 / 103 121 / 115 103 / 111

112 / 125 127 / 160 113 / 123 108 / 111 98 / 107 98 / 111 123 / 125 99 / 123

Group 2 (fast) katG micF d osmY recA uspAd zwf d

100 100 100 100 100 100

105 / 103 97 / 101 99 / 105 106 / 104 107 / 95 108 / 101

109 / 98 111 / 104 103 / 107 99 / 98 100 / 96 105 / 103

95 / 99 117 / 112 104 / 106 101 / 106 109 / 106 99 / 97

93 / 104 119 / 121 111 / 100 110 / 105 110 / 102 107 / 96

103 / 101 108 / 107 118 / 121 105 / 107 108 / 99 108 / 100

90 / 106 114 / 111 122 / 137 101 / 104 101 / 109 103 / 116

99 / 95 117 / 116 138 / 156 109 / 105 102 / 108 105 / 115

95 / 97 109 / 128 156 / 172 113 / 109 108 / 118 108 / 123

a

They are strong alkaline compounds, and medium pH was adjusted to 7.0 with HCl. Promoters are roughly classified into two groups (i.e. fast and slow) by their basal transcriptional activities [24]. c Means of two independent triplicate determinations (n56) are shown here. Although the enzyme activity was corrected by the cell density of each medium, severe growth retardation (.30%) did not occur. Sodium phosphate (Na 2 HPO 4 ) did not affect any promoters even at 5 mM, so the data are omitted. d When the incubation period was prolonged (e.g. .120 min), 500 mM SiW11 induced these promoters up to 130–140% of the control activity. e When the incubation period was prolonged (e.g. .120 min), 500 mM PW11 induced this promoter up to 130–140% of the control activity. b

Y. Tajima / Journal of Inorganic Biochemistry 94 (2003) 155–160

limits various substances penetrating into the cytosol. Findings specific to PW11 are summarized as follows.

3.1.1. DNA-damaging stress-sensitive promoters It is well known that ada, dinB, dinD, nfo, recA and umuDC are DNA-damaging stress-sensitive promoters [24,26–30]. However, PW11 did not significantly induce these promoter genes (Table 1). Some of them were slightly induced by this compound (e.g. 113% recA at 500 mM), but the effects of PW11 on these promoters were weak, and other toxic compounds used as a positive control (e.g. mitomycin C, paraquat) gave much higher scores in general (Table 2). Therefore, we may not come to any conclusions from such a weak response to the compound. In the literature, there is no evidence to suggest that tungsten compounds are carcinogenic or mutagenic [2,3], which is now genetically confirmed by the experimental results shown here. 3.1.2. Oxidative stress-sensitive promoters Interestingly, some polyoxotungstates are highly accumulated in the membrane fraction of the bacterial cell and reduced in vivo [31]. It is well known that the oxidation state of some tungsten atoms in the polyoxotungstate molecule is decreased from W(VI) to W(V) when the compound is reduced [5–9]. Therefore, it is possible that such a reduced polyoxotungstate produces active oxygen species (e.g. superoxide anion (O 2 2 )) by autooxidation of W(V). However, the reported redox potential of PW11 (ca. 21.0 V at around neutral pH) seems to be too low for a biological system to give this molecule an electron [32]. Therefore, the author investigated whether or not PW11 can actually produce active oxygen species in vivo. Table 1 shows that PW11 did not significantly induce katG, soi28 and zwf gene expressions, which are known to be induced by oxidative stress [24,33–35]; for example, they are clearly induced by compounds such as paraquat and H 2 O 2 (Table 2). Furthermore, the amount of H 2 O 2 in

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a medium culturing the bacterial cells (measured by chemiluminescence) was not increased significantly by the addition of tungsten compounds (data not shown). In addition, it has been reported that chronic exposure to dust containing tungsten metal can induce pulmonary fibrosis. However, this does not seem to be due to tungsten itself, but to the toxic effect of active oxygen species generated by interaction with cobalt [36]. These findings suggest that tungsten compounds do not directly produce active oxygen species, i.e. their ability to cause oxidative stress seems to be weak, even present at all.

3.1.3. Osmotic stress-sensitive promoters Curiously, PW11 induced osmY gene expression (Table 1). Usually, this gene is induced by an osmotic signal [37], but the effect of PW11 occurred at a much lower concentration than other ordinary salts, for example, NaCl induced osmY only 2–3-fold at 300 mM (Table 2). Since cationic peptide antibiotics such as polymixin B selectively induced osmY [38], and PW11 is a type of polyanion, it was suggested that osmotic sensor protein related to this promoter gene is sensitive to such a charged substance. Although the detailed mechanism of osmY gene expression has not yet been elucidated, a certain charge interaction may be involved in this system. It is known that the expression of osmY is regulated by an alternate sigma cofactor (s s) of RNA-polymerase, which is translated from the rpoS gene in the stationary phase of the cell cycle [37]. Therefore, it is possible that the osmY gene expression induced by PW11 means simple alteration of growth conditions rather than osmotic stress. However, PW11 did not strongly suppress the bacterial growth (measured by optical density of the culture medium). PW11 is a heptabasic anion with a molecular diameter of ˚ [6–9]. The high negative charge density present in |10 A

Table 2 The effect of various compounds on stress promoters Compound

Concentration

Stress promoter a (Fold induction, %)b

p-Chloroaniline N,N-Dimethylformamide Dimethylsulfoxide Doxorubicin Ethanol HgCl 2 H2O2 Methotrexate Mitomycin C NaCl c NaNO 2 Paraquat 1-Propanol Sucrose

5 mM 10% 10% 25 mM 10% 1 mM 2 mM 25 mM 200 nM 1300 mM 100 mM 50 mM 10% 200 mM

micF (337), osmY (289) clpB (253), micF (279) micF (267) clpB (254), dinB (265), recA (264), umuDC (286) clpB (384), merR (288), micF (325) merR (.1000) dinD (270), katG (294), recA (356), umuDC (295) dinB (267), recA (327), umuDC (290) dinB (265), dinD (286), recA (351), umuDC (292) osmY (255) micF (264), osmY (331) micF (265), nfo (336), soi28 (388), zwf (357) clpB (271), micF (293) osmY (254)

a

When the induction rate was less than 250%, data are omitted in order to minimize this table. Means of two independent triplicate determinations (n56) are shown here. c The culture medium contained salt to some extent, and the additional amount of NaCl is shown here. b

158

Y. Tajima / Journal of Inorganic Biochemistry 94 (2003) 155–160

such a small area is responsible for the strong charge interaction between PW11 and its target molecules, which is hardly affected by steric hindrance. In our previous reports, the salting-in effect and elution power in affinity chromatography of PW11 were 100–1000 fold higher than those of NaCl [39–41]. In addition, it was recently suggested that PW11 may be useful for checking how deeply the charge interaction concerns a certain binding system [42]. Therefore, it is possible that the effect of PW11 on osmY is due to polyanionic characteristic of PW11 . On the other hand, it is well accepted that the micF gene functions as a regulator of porin protein expression (known as the OmpF / OmpC system located in the outer membrane [24,43]). This promoter is known to respond to fluctuations in temperature and osmotic conditions, as well as a wide range of chemicals that interfere with the integrity of the outer membrane (e.g. organic solvents). The membrane permeability of the bacterial cells will be reduced and the transportation across the outer membrane is thus limited under conditions where the micF gene is induced [44]. This is considered to be as a ‘self-defense’ system protecting the bacterial cells from various hazardous substances. As shown in Table 1, the tungsten compounds tested did not significantly induce the micF gene, although it is sensitive to osmotic stress. This is probably because the regulation mechanism of this gene is different with that of the osmY system [44], or at least that is the author’s current hypothesis.

3.1.4. Heavy metal-sensitive promoter and other findings Table 1 shows that neither WO 22 nor PW11 strongly 4 induced merR gene expression, whereas other heavy metals such as Hg 21 induced this gene very strongly (Table 2) [24,45]. This suggests that the mechanism of action of the two tungsten compounds in vivo are distinct from those of other (cationic) heavy metals as noted elsewhere [2,3]. It is well known that the clpB gene product is an analogue of human heat shock protein (HSP70), which responds to agents affecting and / or degenerating various proteins (e.g. organic solvents) [24,46]. The uspA gene is induced under various conditions of growth inhibition, which include nutrient exhaustion as well as the presence of toxic agents (e.g. heavy metals, antibiotics) [24,47]. However, PW11 did not significantly induce these stress promoters. 3.2. Effects of SiW11 on various stress promoters The results are summarized in Table 1. When compared with PW11 , SiW11 induced many stress promoters to a slightly higher extent, and this was more obvious when the incubation time with the polyoxotungstate was prolonged (data not shown). However, except for clpB and osmY, we

may still not come to any conclusions from such relatively weak responses. The molecular structure of SiW11 is almost the same as that of PW11 [6–9]. The differences are that the central tetrahedron of the SiW11 anion is occupied by silicon (Si) instead of phosphorus (P), and its charge is increased from 27 to 28. In addition, the redox potential of SiW11 was reported to be almost equivalent to that of PW11 [32]. Therefore, it is not easy to distinguish between these two compounds using a chemical procedure, but these biological systems (clpB and osmY) revealed the difference. This reason is unclear, but the author considers that this is perhaps because SiW11 has a stronger charge interaction with the target. There is another possibility that PW11 induced the clpB and osmY gene expressions, to a similar extent as SiW11 , but there was a certain (unknown) inhibitory factor specific to the PW11 molecule, thereby lowering the apparent activities of the two promoters.

3.3. Effects of constituents of PW11 /SiW11 Around neutral pH, WO 22 will be partially polymerized 4 (e.g. forming a isopolytungstates such as paratungstate [W7 O 24 ] 62), and ‘Na 2 SiO 3 ’ is a polymeric silicate itself, which undergoes further changes when acidified [1,6]. Therefore, WO 22 and SiO 22 might have some effects on 4 3 the stress promoters similar to PW11 / SiW11 . However, the constituents of PW11 and SiW11 (i.e. 22 22 HPO 22 4 , SiO 3 , WO 4 ) had almost no effect on any promoter gene. In addition, metachromatic reaction of the culture medium was not significant at the given conditions, which means that polyanionic molecular species were not developed in the solution. This is probably because the culture medium contains various organic acids in mM 22 order, and the self-polymerization of WO 22 is then 4 / SiO 3 inhibited. Therefore, we can consider that a chemical feature unique to PW11 / SiW11 and originating from neither of its constituents (i.e. a polyanionic characteristic) plays an important role in the results, as polyoxotungstates gave some positive effects but their constituents did not.

3.4. Effects of PW11 /SiW11 on several representative enzymes It is well accepted that generally the heavy metal compounds are highly toxic and reduce the activities of many enzymes. This is because heavy metal cations (e.g. Hg 21 ) bind with active chemical groups in the enzymes (e.g. hydroxyl group (–OH), sulfhydryl group (–SH)) and block the enzymatic reactions. However, the tungsten compounds tested in this report have an oxoanionic molecular form [1,2], and it seems that these compounds can not directly bind with such a chemical group. Therefore, whether tungsten compounds actually have an inhibitory effect on several representative enzymes was investigated.

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Table 3 Effect of PW11 / SiW11 on various enzyme activities Reagents Enzymes and reactions

None

1 mM WO 22 4 1 1 mM HPO 22 4

1 mM WO 22 4 1 1 mM SiO 22 3

PW11 100 mM

SiW11 100 mM

Restriction enzyme activities a (%) BamHI (pH 8.5)b EcoRI (pH 7.5) HindI (pH 7.5)

100 100 100

88 93 91

84 75 82

76 ,5 ,5

,5 ,5 ,5

Polymerase chain reaction a (%) pH 7.5 pH 8.9 b

100 100

89 90

71 83

,5 54

,5 ,5

Activities of serum enzymes a , c (%) Alanine aminotransferase Amylase Aspartate aminotransferase Cholinesterase Creatine kinase g-Glutamyltranspeptidase Lactate dehydrogenase Leucine aminopeptidase

100 100 100 100 100 100 100 100

98 100 97 101 97 98 99 101

108 106 107 102 101 92 100 106

92 99 93 103 88 98 95 93

91 101 95 104 78 93 96 90

a

All values shown in this table are means of a triplicate determination, and concentrations of reagents shown here are final ones in the reaction mixtures. PW11 and SiW11 will be decomposed from pH 7 and 8, respectively [6]. Therefore, the effect of PW11 will be diminished over pH 8. c In addition to these items, 100 mM PW11 / SiW11 did not interfere with the enzymatic assay systems of urea nitrogen, uric acid, creatinine, cholesterol, triglyceride, bilirubin or glucose (data not shown). b

Table 3 shows that the tested tungsten compounds had almost no effect on many serum enzymes, but PW11 and SiW11 inhibited the activity of enzymes with nucleic acids as their substrates (e.g. restriction enzyme, polymerase), as suggested previously [25]. When the concentration of PW11 / SiW11 was relatively high, the inhibition mode on these enzymes was noncompetitive (|300 mM) or irreversible (|1 mM). However, the inhibition was almost competitive when the concentration of the polyoxotungstates was low (|100 mM) (data not shown). This means that the phenomenon of enzyme inhibition is probably due to polyanionic characteristic of PW11 / SiW11 , which will act as a nucleic acid-analogue. Because nucleic acids can be regarded as a type of polyanion, it is possible that polyoxotungstates inhibit such anion-sensitive enzymes non-specifically as a result of charge interaction. In addition, the DNA-binding activity of anti-DNA antibody was recently found to be inhibited by low concentration of PW11 (|1 mM), which also suggests that PW11 acts as a DNA-analogue [42]. Furthermore, it is generally accepted that many polyoxotungstates such as SiW11 have a potent antiviral activity. This is considered to be due to inhibition of RNA-dependent DNA polymerase [48–50], and / or due to inhibition of the fusion of viral particles to the host cell membrane [51]. In any case, we think that the effects of polyoxotungstates are principally based on their polyanionic characteristics. These findings also seem to support the hypothesis that polyoxotungstates commonly and universally act as nucleic acid-analogues. Herve et al. argued for a similar conclusion [52], i.e. it seems likely that polyoxotungstates also

inhibit several anion-sensitive enzymes non-specifically as a result of the charge interaction by acting as a nucleic acid-analogue.

3.5. Toxicity of tungsten compounds It is well known that molybdenum paucity is induced by chronic administration of WO 22 4 . This seems to be a simple substitution effect because the chemical properties of tungsten are very similar to those of molybdenum [1–3]. Except for this point, it is generally accepted that tungsten compounds such as WO 22 have relatively low 4 toxicity compared with compounds of other heavy metals. This is considered to be principally due to its oxoanionic molecular form which is highly water-soluble, for examples WO 22 is excreted rapidly into urine and is not 4 believed to accumulate in the body, except for organs such as bone and the spleen, even after chronic exposure [2,3]. There have been only a few case reports of acute intoxication due to tungsten compounds [53]. It is known that the umuDC gene is induced under the SOS response, where cell proliferation is interrupted [24,30], but the tungsten compounds tested did not significantly induce this promoter. In addition, the direct toxicity of PW11 / SiW11 to a cultured human cell also seems to be low and their reported growth inhibitory concentration is .100 mM [54]. These findings also support tungsten compounds having a relatively low toxicity. In this report, it was found that tungsten compounds were toxicologically and biologically inert. However, polyoxotungstates do have some biological effects, which

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is probably due to their polyanionic characteristics. This is a unique feature of tungsten compounds when compared with other (cationic) heavy metals.

4. Abbreviations PW11 PW12 SiW11

undeca(11-)tungstophosphate ([PW11 O 39 ] 72) dodeca(12-)tungstophosphate ([PW12 O 40 ] 32) undeca(11-)tungstosilicate ([SiW11 O 39 ] 82)

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