Effects of gamma-ray-induced free radicals on the metal content and amino acid composition of human metallothionein-1.

Metallothioneins (MTs), a low-mass class of metalloproteins, are characterized by a high thiolate sulphur and metal content. MTs are involved in metal homeostasis and heavy metal detoxification, and are efficient scavengers of free radicals. This article describes zinc release from human MT-1 and modification of its amino acid composition when subjected to free radicals generated during gamma ray radiolysis. The effect of gamma ray radiolysis of untreated and metal-depleted human MT-1 was tested under multiple aerobic and anaerobic conditions at increasing irradiation doses. Under all conditions, a rapid increase of serine in the early stages of irradiation was observed. Irradiation for longer times led to cysteic acid formation, except under argon atmosphere. Several other amino acid concentrations gradually decreased. Formation of limited amounts of hydroxyproline, hydroxylysine and ornithine as well as some less common derivatives such as cystathionine occurred as side-effects.

Effects of combined gamma-irradiation and metal (Al+Cd) exposures in Atlantic salmon (Salmo salar L.).

These experiments were designed to investigate transcriptional effects in Atlantic salmon (Salmo salar) after exposure in vivo to ionizing gamma radiation combined with subtoxic levels of aluminum (Al) and cadmium (Cd). Juvenile fish (35 g) in freshwater with or without Al and Cd (255 microg Al/L + 6 microg Cd/L) were exposed to a 75 mGy dose of gamma-irradiation, and induced responses were compared to those of controls. The transcriptional levels of eight genes encoding proteins known to respond to stress in fish were quantified in liver of fish exposed for 5 h to gamma radiation, to Al and Cd or to the combination of Al, Cd and gamma radiation. The studied genes were caspase 3B, caspase 6A, caspase 7, p53 (apoptosis), glutathione reductase (GR), phospholipid hydroperoxide glutathione peroxidase (GSH-Px), (oxidative stress), metallothionein (MT-A) (metal stress) and ubiquitin (Ubi) (protein degradation). The results showed that gamma-irradiation alone induced significant upregulation of caspase 6A, GR, GSH-Px, MT-A and Ubi compared to the control group, while 5 h exposure to Al+Cd alone did not induce any of the studied genes compared to the control. No significant upregulation of the series of investigated genes could be observed in fish exposed to gamma-irradiation in combination with Al+Cl. In conclusion, the results suggest that the presence of Al+Cd in the water counteracted the gamma-irradiation effect by modifying the transcription of genes encoding proteins involved in the defense mechanisms against free radicals in the cells.

Mn,Cd-metallothionein-2: a room temperature magnetic protein.

Naturally occurring metallothionein (MT) is a metal binding protein, which binds to seven Zn2+ through 20 conserved cysteines and forms two metal binding clusters with a Zinc-Blende structure. We demonstrate that the MT, when substituting the Zn2+ ions by Mn2+ and Cd2+, exhibits magnetic hysteresis loop observable by SQUID from 10 to 330 K. The magnetic moment may have originated from the bridging effect of the sulfur atoms between the metal ions that leads to the alignment of the electron spins of the Mn2+ ions inside the clusters. The protein backbone may restrain the net spin moment of Mn2+ ions from thermal fluctuation. The modified magnetic-metallothionein is a novel approach to creating molecular magnets with operating temperatures up to 330 K.

Effects of cadmium and zinc on solar-simulated light-irradiated cells: potential role of zinc-metallothionein in zinc-induced genoprotection.

Zinc is an essential oligoelement for cell growth and cell survival and has been demonstrated to protect cells from oxidative stress induced by UVA or from genotoxic stress due to UVB. In a recent work we demonstrated that the antioxidant role of zinc could be related to its ability to induce metallothioneins (MTs). In this study we identified the mechanism of zinc protection against solar-simulated light (SSL) injury. Cultured human keratinocytes (HaCaT) were used to examine MTs expression and localization in response to solar-simulated radiation. We found translocation to the nucleus, with overexpression of MTs in irradiated cells, a novel observation. The genoprotective effect of zinc was dependent on time and protein synthesis. DNA damage was significantly decreased after 48 h of ZnCl(2) (100 microM) treatment and is inhibited by actinomycin D. ZnCl(2) treatment (100 microM) led to an intense induction, redistribution, and accumulation of MT in the nucleus of irradiated cells. MT expression correlated with the time period of ZnCl(2) treatment. CdCl(2), a potent MT inducer, did not show any genoprotection, although the MTs were expressed in the nucleus. Overall our findings demonstrate that MTs could be a good candidate for explaining the genoprotection mediated by zinc on irradiated cells.

[The survival of mice and the metallothionein content of their livers and kidneys as criteria for assessing exposure to pulsed neutron radiation]. / Vyzhivaemost' myshei i soderzhanie metallotioneinov v ikh pecheni i pochkakh kak kriterii otsenki vozdeistviia impul'snogo neitronnogo izlucheniia.

The effect of pulsed neutron radiation was studied in comparison with continuous neutron radiation and continuous gamma-radiation. Animal survival and induction of metallothionein (MT) synthesis in liver and kidney of mice exposed to equivalent doses were chosen as criteria for evaluation of radiation effects. It was found that the level of MT in liver and kidney of mice exposed to neutron radiation decreased 24 hours after irradiation and then continued decreasing in kidney for 48 hours after irradiation. This is evidence of more intensive free-radical processes initiated by pulsed neutron radiation. At the same time, RBE values of pulsed neutrons did not differ significantly from that of continuous neutron radiation.

Cutaneous metallothionein induction by ultraviolet B irradiation in interleukin-6 null mice.

The mediators of cutaneous metallothionein induction by ultraviolet radiation have not been defined. In this study we sought to identify cytokines that might be involved. We examined the role of interleukin-6, using the IL-6 null (IL-6-/-) mouse, which has been observed to be highly sensitive to ultraviolet radiation damage. Whereas cutaneous metallothionein concentration, measured by radioimmunoassay, began to rise in wild-type (IL-6+/+) mice by 12 h after ultraviolet irradiation, there was a significant delay in the IL-6-/- mice until 48 h after UV irradiation. Immunohistologically, metallothionein appeared in IL-6+/+ mice at 24 h in dermal fibroblasts, and then by 48 h in epidermal basal keratinocytes, with intensity increasing until 72 h, and was coincident with proliferating cell nuclear antigen-positive staining. Corresponding metallothionein expression in IL-6-/- mouse skin was significantly delayed. Serum interleukin-6 was elevated in IL-6+/+ mice following ultraviolet irradiation, with peak concentration at 4 h, but no increase in serum interleukin-1beta was found in either IL-6+/+ or IL-6-/- mice. Interestingly, tumor necrosis factor alpha concentration in serum was elevated at 12 h postirradiation in IL-6+/+ mice, but there was an earlier (at 4 and 8 h) time-dependent increase in tumor necrosis factor alpha in serum of the IL-6-/- mice. Skin zinc and copper concentrations were not altered by ultraviolet irradiation in either IL-6+/+ or IL-6-/- mice. The results suggest that interleukin-6 may be a very early mediator of cutaneous metallothionein induction by ultraviolet radiation, but that this role is possibly assumed by alternative cytokines like tumor necrosis factor alpha when interleukin-6 is deficient.

[The diverse effects of the combined action of cadmium chloride and ionizing radiation on the content of metallothioneins in mouse bone marrow and liver]. / Razlichnye éffekty sochetannogo deistviia khloristogo kadmiia i ioniziruiushchei radiatsii na soderzhanie metallotioneinov v kostnom mozge i pecheni myshei.

It was shown that introduction of cadmium chloride (0.75 mg/kg Cd2+) in combination with gamma irradiation (8.5 Gy) of mice increases the level of metallothioneines (MTs) in the bone marrow and liver of mice. The maximum effect was observed in 24-30 h after the performance. Similarly, irradiation with the doses of 3 to 10 Gy resulted in an increase in the contents of both MT isoforms (MT1 and MT2) in the bone marrow in 24 h. A combined action of gamma irradiation and a heavy metal caused an additive effect on the MT content in the bone marrow, whereas the MT content in liver was 2 times lower than that predicted theoretically. A possible mechanism of the discovered phenomenon was discussed. Supposedly, it is associated with different degrees of the radiation-induced inhibition of the MT1 and MT2 expression by cadmium ions.

Stable thiyl radicals in dried yeast Cu(I)6-thionein.

It was of interest to obtain long-lived thiyl radicals embedded in organic matrices. Solid thiol compounds including penicillamine, glutathione, and cysteine were UV irradiated under anaerobic conditions at 293 K for 60 min. The formed radicals were identified by electron paramagnetic resonance (EPR) (g = 2.0265 +/- 0.0015) at 293 K as thiyl radicals. The blue-colored radical species were subjected to reflection spectrometry (lambda max = 601 +/- 3 nm). The color and the EPR signal remained unchanged for six months. At the same time, the UV irradiation of lyophilisized yeast Cu(I)6-thionein generated stable EPR detectable thiyl was seen when the Cu(I)-thiolate was used. No EPR detectable thiyl radicals radicals at a g-value of 2.026 +/- 0.001. Unlike irradiated cysteine, a five times higher concentration of thiyl radicals were measured in the Cu(I)-thiolates of penicillamine, glutathione, and thiophenole, indicating that the hexanuclear copper arrangement in Cu(I)-thionein is most suitable for both the formation and stabilization of this sulfur radical species.

Induction of metallothionein synthesis in transplanted murine tumors by X irradiation.

Although recent studies have shown that radiation can induce metallothionein (MT) synthesis in normal tissues, the induction of tumor MT synthesis by irradiation has not been reported. We examined the accumulation of MT in the Meth-A tumor (mouse fibrosarcoma cells) transplanted into mice exposed to whole-body X irradiation. In the present study, the MT content in the tumor cells was increased by X irradiation in a dose-dependent manner. The MT level induced in the tumor cells by X irradiation was elevated not only after a single exposure but also after repeated exposures. Several studies have shown that MT is one of the important cellular factors in resistance to various anticancer drugs and ionizing radiation. Thus our results suggest that the radiation-induced MT in the tumor cells may have to be taken into consideration when designing protocols for radio- and chemotherapy.

[The property of radiation in low doses and of low-intensity ionizing radiation to cause metallothionein induction]. / Svoistvo radiatsii v malykh dozakh i nizkointensivnogo ioniziruiushchego izlucheniia vyzyvat' induktsiiu metallotioneinov.

The effect of the increase in metallothionein content in hepatocytes of white mice located in conditions of chronic low dose rate and low-dose irradiation. In this work an opportunity of participation of metallothioneins, as the natural radioprotector and factor of nonspecific resistance, in adaptation of organisms to adverse environmental conditions was discussed.

[The radiobiology of metallothioneins]. / Radiobiologiia metallotioneinov.

The data about the increase of cell and tissue metallothioneins (MT) level after the irradiation by ionizing radiation and UV rays were present. The conclusion was made that increase was conditioned by DNA damage by free radicals with following MT-genes amplification. Some agents which increased endogenous MT content also exogenous MT protected biological object from the irradiation and agents which caused oxidative stress. The possible mechanisms of MT protection effect (principal is the MT antioxidative properties) and some aspects of practical application of MT were examined.

Radiolysis of metallothionein in deaerated and oxygen-saturated solutions.

Gamma radiolysis of metallothionein (MT) in phosphate-buffered solutions at pH 8.0 leads to the formation of intra- and intermolecular disulfide bridges as well as the release of chelated metal ions. When MT solutions deaerated with N2 or saturated with N2O were irradiated, G values for RS- loss as determined by Ellman's reagent were 4.2 and 7.0, respectively, whereas those for dimerization were only 0.32 and 0.67. A short chain reaction existed in the decrease in RS- when radiolysis was performed in O2-saturated solutions. The rate constants for reactions of MTs with OH radical and H2O2 were measured to be about 10(12) and 100 M-1 s-1, respectively. A mechanism for the radiolysis of MT-1 is proposed.

[The content of metallothioneins in mouse bone marrow and liver and in human lymphocytes after whole-body gamma irradiation]. / Soderzhanie metallotioneinov v kostnom mozge i pecheni myshei i v limfotsitakh cheloveka pose obshchego gamma-oblucheniia.

The increase in the content of bone marrow and hepatic metallothioneins (MT) in mice with the maximum at 30 hr after whole-body gamma irradiation was shown. The MT level in that tissues at that time correlated with the exposure dose. The MT content in lymphocytes of patient with acute lympholeucosis was increased after fractionated whole-body irradiation, that index also correlated with accumulated exposure dose.

[The connection between the content of metallothioneins in the bone marrow and liver and the survivability of irradiated mice after cadmium chloride administration]. / Sviaz' mezhdu soderzhaniem metallotioneinov v kostnom mozge, pecheni i vyzhivaemost'iu obluchennykh myshei posle vvedeniia khloristogo kadmiia.

The survival rate of mice gamma-irradiated at different times after administration of Cd2+ (0.75 mg/kg) was shown to be tightly connected with the amount of metallothioneins induced by this heavy metal in the bone marrow (r = 0.96). The connection between the amount of metallothioneins in the liver and the survival rate was less pronounced.

Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals.

Rabbit liver metallothionein-1 (Mr 6500), which contains zinc and/or cadmium ions, appears to scavenge free hydroxyl (.OH) and superoxide (O-.2) radicals produced by the xanthine/xanthine oxidase reaction much more effectively than bovine serum albumin (Mr 65 000) which was used as a control. Kinetic competition studies between metallothionein and either a spin trap for .OH or ferricytochrome c for O-.2 radicals, gave bimolecular rate constants of the order of kOH/MT approximately equal to 10(12) M-1 X s-1 and kO-2/MT approximately equal to 5 X 10(5) M-1 X s-1, respectively. The former value suggests that all 20 cysteine sulfur atoms are involved in this quenching process and that they all act in the diffusion control limit. The aerobic radiolysis of an aqueous solution of metallothionein, generating O-.2 and .OH radicals, induced metal ion loss and thiolate oxidation. These effects could be reversed by incubation of the irradiated protein with reduced glutathione and the appropriate bivalent metal ion. Metallothionein appears to be an extraordinarily efficient .OH radical scavenger even when compared to proteins 10-50-times its molecular weight. Moreover, hydroxyl radical damage to metallothionein appears to occur at the metal-thiolate clusters, which may be repaired in the cell by reduced glutathione. Metallothionein has the characteristics of a sacrificial but renewable cellular target for .OH-mediated cellular damage.