Induction of cell-proliferation hormesis and cell-survival adaptive response in mouse hematopoietic cells by whole-body low-dose radiation.

Hormesis and a cytogenetic adaptive response induced by low-dose radiation (LDR) have been extensively documented. However, few studies have investigated the induction of an adaptive response by LDR for cell survival in vitro. In the present study, we investigated whether LDR could induce hormesis in hematopoietic cells and the adaptive response of these cells to subsequent high-dose radiation-induced cytotoxic effects. Mice were exposed in whole-body to 0 (as control), 0.05, 0.25, 0.50, 0.75, and 1.00 Gy of X-rays. They were killed 12, 24, 48, and 72 h later to observe the stimulating effect of LDR on total bone marrow cells per femur and bone marrow progenitor, colony-forming unit-granulocyte-macrophage (CFU-GM). Exposure to 0.5 Gy of X-rays resulted in significantly stimulating effects on both parameters with a maximum effect at 48 h, showing a cell-proliferation hormesis. In the next experiment, mice were irradiated by 0.5 Gy X-rays as an adaptive exposure (D1), and 6, 12, 24, 48, and 72 h later, they were exposed to 6 Gy X-rays as a challenging exposure (D2). Forty-eight h after D2, cytotoxic effects were analyzed using peripheral blood cells (red blood cells, white blood cells, and platelets) and bone marrow cells (total bone marrow cells of the femur, and bone marrow progenitors such as CFU-GM and erythroid burst-forming unit, BFU-E). An adaptive response to D2-induced cytotoxic effect, named as the cell-survival adaptive response, was found in both peripheral blood cells and bone marrow cells when D1 and D2 exposures were given at intervals of 24-48 h. These results suggested that LDR could induce both cell-proliferation hormesis and cell-survival adaptive response to subsequent high-dose radiation in bone marrow cells. It may be of potential importance, if this phenomenon is confirmed clinically, since it may be applied to reduce the adverse effect of radiotherapy.     

Induction of immune tolerance by oral IVIG

In the last years evidence has been provided for the importance of B cells in the pathogenesis of rheumatoid arthritis (RA). Several studies have supported the concept that humoral immunity, manifested by the production of autoantibodies, such as rheumatoid factors (RFs), plays a significant role in the course of the disease. Specific targeting of autoantibody-producing B cells, such as RF-producing B cells, should therefore be a promising new approach in the treatment of RA. We used a mouse model to induce human RF responses and asked the question whether oral treatment with the antigen (human IgG) recognized by RFs could induce immune tolerance to RF responses. Balb/c mice were orally treated with polyvalent human IgG before and after immunization with insoluble immune complexes (ICs) that triggered the induction of RFs. Serum titers of RFs were significantly reduced after both primary and booster immunization when human IgG was given as a single oral dose or continuously in drinking water. Continuous treatment with human IgG even prevented booster effects on RFs when treatment started after primary immunization. Treatment with IgG fragments provided evidence that the observed effect of human IgG was mediated by the Fc part and not the Fab part of IgG. Furthermore, transfer of spleen cells obtained from mice after oral treatment with human IgG suppressed RF responses in recipient mice. These data give promising indications that oral human IgG might represent an alternative approach for immunosuppressive B-cell targeted therapies in RA.     

Radioprotective effects of exogenous glutathione against whole-body gamma-ray irradiation: age- and gender-related changes in malondialdehyde levels,superoxide dismutase and catalase activities in rat liver

Age- and gender-related changes in malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) activities in rat livers exposed to different doses of whole-body gamma-ray radiation were determined. In addition, the effects of exogenous glutathione (GSH) against radiation injury in rat livers were investigated. We found that MDA levels have an age-associated increment and an increasing radiation dose-related elevation, although they decrease slightly in the 4 Gy group. The MDA levels in old rats were lower in males than in females, while those of young rats did not change. There were no observed age-related changes in SOD activities, although male rats had higher SOD activity than females. Female rats had the highest CAT activities in the 4 Gy group, while male rats had the highest CAT activities in the 6 Gy group. CAT activities in the 8 Gy group were lower than those of the 2 Gy group for each gender and age. While MDA levels were decreased and CAT activities increased by GSH, SOD activities remained unchanged. The results indicate that gamma-ray radiation affects gender- and age-dependent MDA levels, SOD and CAT activities. Administration of GSH appears to be a useful approach to reduce radiation injury by reducing MDA levels and increasing CAT activities.     

Induction of mouse liver glutathione S-transferase by ethanol

The induction of hepatic glutathione S-transferase by ethanol was investigated in male Swiss-Webster mice using a liquid diet. After a 7-day feeding period, mice that received 18, 27 or 36% of their calories as ethanol exhibited significant increases in the specific activity of glutathione S-transferase when 1,2-dichloro-4-nitrobenzene (DCNB), p-nitrobenzylchloride (NEC) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENP) were used as substrates. The observed increases in activity appeared to be related to the concentration of ethanol in the diet. Thus, mice fed a diet with 36% of the calories as ethanol exhibited the greatest increase in specific activity (DCNB, 75%; NBC, 60%, ENP, 34%). Pair-fed mice demonstrated similar changes in enzymatic activity. A time-course study indicated a 4-day feeding period was not sufficient to elicit significant induction, but a significant increase was apparent by day 7. This increase was maintained or increased through day 14. By comparison, 0.5 mg of phenobarbital/ml of diet produced a greater increase in enzymatic activity (DCNB, 449%; NBC, 227%; ENP, 219%). These results suggest that ethanol does induce glutathione S-transferase, but it is a relatively poor inducer of this enzyme.     

Induction of rat hepatic and intestinal glutathione S-transferases by dietary butylated hydroxyanisole.

To obtain insight into the protection mechanism of butylated hydroxyanisole (BHA), a widely used food preservative with anticarcinogenic properties, we investigated the effects of dietary BHA on rat hepatic and intestinal glutathione S-transferase (GST) enzyme activity, and GST isozyme levels. In the proximal small intestine and liver, BHA supplementation significantly increased GST enzyme activity as compared with controls (2.3- and 1.7-fold, respectively, P less than 0.05). GST class alpha and mu contents were significantly higher only in the small intestine (1.6-2.1-fold and 1.3-1.5-fold, respectively, P less than 0.05), whereas GST class pi was significantly induced in liver (4.6-fold, P less than 0.05).     

Induction of quinone reductase and glutathione transferase in rat tissues by juglone and plumbagin

The ability of the naturally-occurring naphthoquinone derivatives, juglone and plumbagin, to increase tissue activities of the Phase II detoxification enzymes quinone reductase (QR) and glutathione transferase (GT) has been investigated in rats. Groups of female Sprague-Dawley rats were dosed by oral intubation on 5 consecutive days with either juglone or plumbagin at 12.5, 25, 50, 75, 100 or 125 mumoles/kg/day. The animals were then killed and the activities of QR and GT determined in tissue homogenates. The naphthoquinone derivatives had no significant effect on enzyme activities in the liver, spleen, heart, lung or urinary bladder. Increases in the activities of one or both enzymes were recorded, however, in the caecum, kidney, forestomach, duodenum, colon, glandular stomach and jejunum. The possibility that induction of Phase II enzymes could contribute to the previously-reported ability of juglone and plumbagin to protect animals against chemically-induced intestinal neoplasia is discussed.     

Induction of rat UDP-glucuronosyltransferase and glutathione S-transferase activities by L-buthionine-S,R-sulfoximine without induction of cytochrome P-450

The effect of prolonged exposure to buthionine sulfoximine (BSO) on rat hepatic Phase I and Phase II drug-metabolizing enzymes has been examined. Exposure to 30 mM BSO in drinking water for 7 days induced hepatic microsomal UDP-glucuronosyltransferase activity (detergent-activated) toward p-nitrophenol (250%), 1-naphthol (210%), morphine (130%) and testosterone (140%), but not estrone. Glucuronosyltransferase activities were also induced after exposure for as short as 3 and as long as 13 days. When rats were returned to unsupplemented drinking water for 1 day prior to sacrifice following 6 days on 30 mM BSO, comparable induction to that seen after 7 consecutive days on the BSO solution was observed despite liver glutathione concentration having rebounded to 127% of control. Daily ingestion of BSO was similar (1 mmol/rat/day) for all periods of 30 mM BSO-drinking water exposure, with a body weight-adjusted dose range of 3.2-6.3 mmol/kg/day. An analogous inductive response caused by drinking 30 mM BSO for 3 days was elicited for p-nitrophenol and morphine glucuronidation by 6 mmol/kg doses of BSO given as single daily intraperitoneal or intragastric injections for 3 days. Intraperitoneal, intragastric and all BSO-drinking water exposures also significantly induced (130-195%) cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene. Significant increases in UDP-glucuronosyltransferase and glutathione S-transferase activities were also observed following 3 days of exposure to BSO in the drinking water at a concentration as low as 5 mM. Cytosolic p-nitrophenol sulfotransferase activity, with one minor exception, was not enhanced by any BSO treatment regimen. Alterations in transferase activities were not accompanied by any major changes in either overall cytochrome P-450 concentration or oxidative reactions selective for two isozymes. Thus, in addition to its well-documented glutathione-depleting property, BSO also selectively induces several Phase II drug-metabolizing enzymes, an effect to be considered in studies employing extended BSO treatment.     

Improvement of murine immune functions in vitro by thioproline

Previous studies have shown that several immune functions were improved in mice after the ingestion of a thioproline (thiazolidine-4-carboxylic acid) enriched diet. In the present work, we have studied the in vitro effects of several concentrations of this thiol compound (0.1, 0.5, 1, 2.5 and 5 mM) on the most relevant functions of three pivotal immune cells, namely, macrophages, lymphocytes, and natural killer (NK) cells from BALB/c mice. The results show that thioproline stimulates the phagocytic process of macrophages, increasing the mobility directed to the inflammatory focus (chemotaxis) and the phagocytosis of inert particles. It increases the adherence and the chemotaxis capacities of lymphocytes, their proliferative activity and favours the natural cytotoxic activity that could improve the capacity to destroy malignant cells. Thioproline concentrations of 0.5 and 1 mM were the most effective regarding the different functions analysed. These results suggest that the improvement of immune functions, observed in previous work, after thioproline-enriched diet ingestion is due to a direct action of this thiol compound on immune cells.     

Radiobiological basis of low-dose irradiation in prevention and therapy of cancer

Antimutagenic DNA damage-control is the central component of the homeostatic control essential for survival. Over eons of time, this complex DNA damage-control system evolved to control the vast number of DNA alterations produced by reactive oxygen species (ROS), generated principally by leakage of free radicals from mitochondrial metabolism of oxygen. Aging, mortality and cancer mortality are generally accepted to be associated with stem cell accumulation of permanent alterations of DNA, i.e., the accumulation of mutations. In a young adult, living in a low LET background of 0.1 cGy/y, the antimutagenic system of prevention, repair and removal of DNA alterations reduces about one million DNA alterations/cell/d to about one mutation/cell/d. DNA alterations from background radiation produce about one additional mutation per 10 million cells/d. As mutations accumulate and gradually degrade the antimutagenic system, aging progresses at an increasing rate, mortality increases correspondingly, and cancer increases at about the fourth power of age. During the past three decades, genomic, cellular, animal and human data have shown that low-dose ionizing radiation, including acute doses up to 30 cGy, stimulates each component of the homeostatic antimutagenic control system of antioxidant prevention, enzymatic repair, and immunologic and apoptotic removal of DNA alterations. On the other hand, high-dose ionizing radiation suppresses each of these antimutagenic protective components. Populations living in high background radiation areas and nuclear workers with increased radiation exposure show lower mortality and decreased cancer mortality than the corresponding populations living in low background radiation areas and nuclear workers without increased radiation exposure. Both studies of cancer in animals and clinical trials of patients with cancer also show, with high statistical confidence, the beneficial effects of low-dose radiation.     

Formation and mass spectrometric analysis of DNA and nucleoside adducts by S-(1-acetoxymethyl)glutathione and by glutathione S-transferase-mediated activation of dihalomethanes

The dihalomethane CH(2)Cl(2) is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA-dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH(2)OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH(2)OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A > C. The four DNA nucleosides were also reacted with the dihalomethanes CH(2)Cl(2) and CH(2)Br(2) in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH(2)OAc and DNA. CH(2)Cl(2) and CH(2)Br(2) yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH(2)OAc model system.     

Role of metabolic activation, covalent binding, and glutathione depletion in pulmonary toxicity produced by an impurity of malathion

O,O,S-Trimethyl phosphorothioate (OOS-TMP) is an impurity present in widely used organophosphorus insecticides such as malathion. Oral treatment of rats with the compound produces prominent bronchiolar epithelial necrosis. Following the administration of [3H]OOS-TMP to rats, substantial amounts of radiolabeled material were covalently bound to lung with a concomitant depletion of glutathione (GSH). Other organs showing significant covalently bound radioactivity were liver, kidneys, and ileum. The maximal accumulation occurred in the tissues within 6 hr, and reached a plateau between 6 and 12 hr. Pretreatment of rats with either phenobarbital or piperonyl butoxide decreased the level of radiolabeled material bound in lung, GSH depletion, and the toxicity of OOS-TMP. These results suggest that the covalent binding is due to a metabolite(s) of OOS-TMP and that the metabolite(s) is involved in the mechanism of toxicity of OOS-TMP. Pulmonary GSH may play a protective role against OOS-TMP induced lung toxicity.     

Modulation of inflammatory immune reactions by low-dose ionizing radiation: molecular mechanisms and clinical application

During the last decade, a multitude of experimental evidence has accumulated showing that low-dose radiation therapy (single dose 0.5-1 Gy) functionally modulates a variety of inflammatory processes and cellular compounds including endothelial (EC), mononuclear (PBMC) and polymorphonuclear (PMN) cells, respectively. These modulations comprise a hampered leukocyte adhesion to EC, induction of apoptosis, a reduced activity of the inducible nitric oxide synthase, and a lowered oxidative burst in macrophages. Moreover, irradiation with a single dose between 0.5-0.7 Gy has been shown to induce the expression of X-chromosome linked inhibitor of apoptosis and transforming growth factor beta 1, to reduce the expression of E-selectin and L-selectin from EC and PBMC, and to hamper secretion of Interleukin-1, or chemokine CCL20 from macrophages and PMN. Notably, a common feature of most of these responses is that they display discontinuous or biphasic dose dependencies, shared with "non-targeted" effects of low-dose irradiation exposure like the bystander response and hyper-radiosensitivity. Thus, the purpose of the present review is to discuss recent developments in the understanding of low-dose irradiation immune modulating properties with special emphasis on discontinuous dose response relationships.     

Ovalbumin modified by gamma irradiation alters its immunological functions and allergic responses

It is well known that gamma (gamma)-ray irradiation results in the alteration of biological function of bioactive materials such as proteins, saccharides and lipids. In this study the effect of gamma-irradiation on the chemical and immunological property of an allergen, ovalbumin (OVA), was investigated. Irradiation of more than 10 kGy resulted in the alteration of the structure of OVA. However, OVA treated with 10 kGy irradiation (10 kGy-OVA), but not 100 kGy-OVA, fully maintained immunological reactivity to a monoclonal antibody specific to the intact allergen (clone 14). Mice immunized with 10 kGy- as well as 100 kGy-OVA showed significantly lower antibody response to the allergen than those with intact OVA in a gamma-ray dosage-dependent manner. Especially immunization of both 10 kGy- and 100 kGy-OVA induced a significant decrease of OVA-specific IgE. Splenocytes of mice immunized with irradiated OVA showed a significant reduction in OVA-specific T cell proliferation and the secretion of Th1-type (IFN-gamma and IL-2) and Th2-type cytokines (IL-4 and IL-6). The expression of T cell activation markers such as CD25 and CD44 was also down-regulated in T cells of mice immunized with irradiated OVAs. These results suggest that gamma-ray irradiation of OVA suppress humoral and cellular immune responses specific to the allergen OVA, and the modification method with gamma-irradiation may be available for the control of allergy.