Respuesta de linfocitos humanos ante Pequeñas dosis de radiación gamma

Linfocitos humanos fueron irradiados en un campo de radiación gamma de baja intensidad para determinar la expresión de las proteínas de choque calórico en función de la dosis. Los linfocitos fueron obtenidos de individuos cuyo trabajo los identifica como ocupacionalmente expuesto y no ocupacionalmente expuestos. La identidad de las proteínas se realizó utilizando anticuerpos contra las proteínas Hsp25, Hsp60, Hsp70 y Hsp90. De éstas, solamente la proteína Hsp70 fue detectada antes y después de la irradiación. Los linfocitos del personal ocupacionalmente expuesto y no ocupacionalmente expuesto expresaron, antes y después de la irradiación, solamente la proteína Hsp70. La cantidad de proteína resultó directamente proporcional al tiempo de irradiación. Después de una dosis gamma de 70.5 mGy, los linfocitos del individuo ocupacionalmente expuesto expresaron una mayor cantidad de proteína Hsp70 que la expresada por los linfocitos del personal no ocupacionalmente expuesto. Este hecho es indicio de que el individuo ocupacionalmente expuesto tiene una mayor tolearancia a los rayos gamma (gamma-tolerancia), inducida por un proceso de adaptación generada por su condición laboral.

ABSTRACT:

Radiation and non-radiation workers lymphocytes were exposed to a low strength gamma-ray field to determine heat shock protein expression in function of radiation dose. Protein identification was carried out using mAb raised against Hsp25, Hsp60, Hsp70 and Hsp90; from these, only Hsp70 protein was detected before and after lymphocyte irradiation. In all cases, an increasing trend of relative amounts of Hsp70 in function to irradiation time was observed. After 70.5 mGy gamma-ray dose, radiation worker's lymphocytes expressed more Hsp70 protein, than non-radiation workers' lymphocytes, indicating a larger tolerance to gamma rays (gamma tolerance), due to an adaptation process developed by their labor condition.

Key words: Key words: Adaptation, Gamma-rays, Heat Shock Proteins, Response, Lymphocytes.

One of the Radiation Biology tasks is to develop tests to indicate exposure to natural or artificial, ionizing radiation fields. Some of these tests use biological indicators (biodosimeters), which correlate the exposure levels with the biological effect.

Molecular chaperons, including the heat-shock proteins, Hsp, are a ubiquitous feature of eukaryotic and prokaryotic cells [1]. Hsp are diverse in size and oligomeric composition, yet several have a common functional theme-modulating the folding and unfolding of other proteins and faciliting assembly and disassembly of multisubunit complexes [2]. Hsp are a family of proteins expressed in response to a wide range of biotic and abiotic stressors to protect the cell from the damage produced by the stress [3]. Due to their extraordinary high degree of identity at the amino acid sequence level and because this cellular stress response has been described in nearly all organism studied, this group of proteins is unique [1].

Anoxia, heat, ethanol, oxygen peroxide, heavy metals, arsenicals, UV radiation, low frequency electromagnetic fields and intensive gamma-ray radiation fields are known as cell stressors that promote Hsp expression [4-7]. This has led to the recognition that Hsp protect cells from many forms of stress through chaperoning effects on protein [8].

Low doses of ionizing radiation, or chemical agents, could induce mechanisms whereby cells become better fit to cope with subsequent exposures to high doses [6]. It has been shown that Hsp70 plays a central role in stress preconditioning, where Hsp induction correlates with protection from subsequent injury [9]. In most organisms, Hsp70 are among prominent proteins induced by heat [10]. There is a close correlation between the induction of these proteins and the induction of tolerance to high temperatures [2,11].

Species thresholds for Hsp expression are correlated with levels of stress that they naturally undergo. Due to its responsiveness to diverse forms of stress, the heat-shock response has undergone widespread applications in biomonitoring and environmental toxicology [7].

Cells of radiosensitive mammalian cell lines usually have a mean lethal dose of about 0.5 Gy, and the majority of them have a defined DNA repair defect. However, defects in DNA repair enzymes alone are not sufficient to explain the range of cellular responses to ionizing radiation. There are normal cells that are very radiosensitive, such as some peripheral body lymphocytes [5]. Ionizing radiation produces denatured proteins via direct ionization as well as by reaction with radiolysis products, enhancing Hsp 70 synthesis in Chinese hamster ovary cells exposed to 400 or 1000 Gy [12]. However there is an interest to determine cell response at low doses of gamma rays [13], and to search for a cellular indicator of ionizing radiation exposure [14, 15].

The aim of this research was twofold: To determine Hsp expression in human lymphocytes irradiated by low strength gamma-ray field, and to observe if there was a response difference between radiation worker, RW, and non-radiation worker, NRW, lymphocytes.

Lymphocytes were collected from RW and NRWs blood samples. Lymphocytes were irradiated at four irradiation times to determine the Hsp expression in terms of radiation dose. Protein identification was carried out using mAb raised against Hsp25, Hsp60, Hsp70 and Hsp90. We found that Hsp70 was only expressed by all sets of lymphocytes, before and after radiation treatment. An increasing trend of relative amounts of Hsp70 protein in function to irradiation time was observed. Radiation worker´s lymphocytes enhanced Hsp70 expression for radiation doses larger than 70.5 mGy, indicating a larger tolerance to gamma rays (gamma tolerance), due to adaptation process developed by their labor condition.

MATERIALS AND METHODS

Subjects

Blood samples from four healthy male individuals were used to isolate their lymphocytes. The individuals have similar life style standards except their jobs. One is 38 years old and has been RW for 11 years, his labor condition keep him exposed at approximately 0.94 mSv-h^-1, due to gamma radiation; the others (NRWs) are 44, 45, and 46 years old, they are only exposed to background radiation, 0.24 mSv-h^-1 due to gamma radiation. All individuals do not smoke, neither consume alcohol.

Lymphocytes collection and culture

From each subject, blood samples were taken in heparinized tubes. Lymphocytes were isolated using gradient of Ficoll-Histopaque (Sigma Chemical Co, St Louis MO, USA, 1077-1), as described by Boyum [16]. From each subject, lymphocytes were cultured in RPMI medium (Gibco BRL, Grand Island NY, USA, 11876-026), supplemented with 100 U/ml of penicillin, 100 ng/ml of streptomycin (In Vitro, Mexico city, Mexico), 0.08 U/ml of insulin (Eli Lilly Mexico city, Mexico), and 5% fetal bovine serum (Gibco BRL, Grand Island, NY, USA, 16000-044). From each subject, lymphocytes viability was estimated by tripan blue exclusion (Sigma Chemical Co, St Louis MO, USA, T-6146).

Biosynthetic labeling and cells irradiation

From each subject, lymphocytes were divided into five aliquot. One aliquot was taken as control to determine the basal levels of Hsp. The other four aliquot were labeled with ³⁵S-methionine (Amersham Laboratories, Buckinghamshire, England, UB2688), whose specific activity was 5 mCi/ml. Then were exposed to gamma radiation field produced by two ¹³⁷Cs gamma ray sources (DuPont Radiopharmaceutical Div. N. Billerica, MA, USA), whose activities are 3.7 x 10⁴ Bq and 1.6 x 10⁵ Bq. At the irradiation site both sources and background radiation produce a dose rate in air of 141 mGy-h^-1. The exposure times were 15, 30, 45 and 60 minutes, meaning a total dose of 35.2, 70.5, 105.7 and 141 mGy respectively. After each irradiation treatment, lymphocyte viability was measured.

Cell Lysis

After irradiation, lymphocytes from all aliquot were centrifuged at 326 g for 10 minutes, and washed twice with 1 ml of phosphate-buffered saline (PBS; pH 7.2) (Gibco BRL, Grand Island NY, USA, 21300-058). Lymphocytes were homogenized with lysis buffer containing 1% triton X-100, 150 mM NaCl, 50 mM tris-HCL (pH 8.0), 5 mM EDTA and 1 mM protease inhibitor, PMSF (Sigma Chemical Co, St Louis MO, USA). The lysate was centrifuged at 1600 g for 10 minutes and supernatant was recovered.

SDS-PAGE and immunoblotting analysis

Total protein quantification was carried out following the Bradford's procedure [17]. From each experimental condition, 30 mg of protein was separated using SDS-PAGE (10% acrylamide) following the technique described by Laemmli [18]. For Western blot, the proteins were blotted onto nitrocellulose membrane (Amersham Laboratories, Buckinghamshire, England, RPN303C), as described by Towbin et al. [19]. After, blocking with 3% skim milk powder (Compañia Nestle, SA de CV, Mexico DF, Mexico) in PBS for 12 hours. Blotts were probed with mAb raised against 25, 60, 70 and 90 Hsp (Sigma Chemical Co, St Louis MO, USA, H-0148, H-4149, H-5147, H-1775) diluted 1:1000 in 3% skim milk powder/PBS for 1 hour. Antibodies binding was visualized by horseradish peroxidase conjugated anti mouse IgG (Sigma Chemical Co, St Louis MO, USA, A-9044) diluted 1:1500 in 3% skim milk. Peroxidase was catalyzed by enhanced chemiluminescence Western blotting detection reagent, ECL (Amersham Laboratories, Buckinghamshire, England, RPN2106).

Autoradiography and densitometry

Nitrocelulose membrane was exposed to a radiographic film BioMax (Eastman Kodak Co, Rochester, NY, USA, 870-1302) for 45 days. Controls autoradiographies, ³⁵S-labeled and ECL revealed immunoblot were read using optic densitometry (Eagle Eye, Estategene Mitsubishi) to determine the amount of newly sintetized specific protein through the³⁵S to ECL ratios.

RESULTS

Cell Viability

Before and after lymphocytes irradiation viability was larger than 90%, this implies no cell death due to gamma rays.

Expression of stress protein in human lymphocytes due to gamma ray radiation field

The expression of several Hsp members in RW and NRWs lymphocytes was analyzed using ³⁵S-methionine as label and irradiated by a low strength gamma-ray field at 37 ^oC, using four different times, 15, 30, 45 and 60 minutes. Lymphocytes lysate was separated on 10% SDS-PAGE and transferred to nitrocellulose membrane. Membrane was sequentially probed with monoclonal anti-Hsp25, Hsp60, Hsp70, Hsp90 and a peroxidase conjugated antimouse IgG antibody. The ECL and radiolabeled Hsp form were visualized by autoradiography. From the antibodies used, Hsp70 was identified in lymphocytes from RW and NRWs (a, b, c) before and after the irradiation, this is shown in Figures 1 and 2.

Gamma-ray field increases Hsp70 expression in human lymphocytes

Western blot-ECL and ³⁵S autoradiography were evaluated by densitometry to determine the amount of newly synthesized specific protein in lymphocytes exposed to low strength gamma-ray field. For each irradiation time, the ³⁵S to ECL ratios of optical densitometry readings were calculated to determine the new synthesized Hsp70 protein. In Figure 3 this ratios for RW and NRWs lymphocytes in function of irradiation times are shown. Here a trend of increased relative Hsp70 protein, in function to irradiation times is observed. Figure 3 shows approximately the same response for lymphocytes exposed to 35.2 and 70.5 mGy gamma-ray doses. After 70.5 mGy gamma dose RW lymphocytes expressed larger amounts of Hsp70 in comparison to NRWs lymphocytes.

The aim of this research was twofold: To determine Hsp expression in human lymphocytes irradiated by low strength gamma-ray field, and to observe if there was a response difference between a radiation worker and non-radiation workers lymphocytes. When human lymphocytes are irradiated with low strength gamma ray field Hsp70 protein expression is enhanced. This response has been observed in other types of cells exposed to more intense radiation fields [12], and other stressors, as heat and hydrogen peroxide [7], that leads to the rapid and transient activation of genes encoding heat shock proteins [20].

Low intensity gamma-ray radiation field stress human lymphocytes; the probable explanation is the following: During gamma ray interaction with water free radicals, such as hydrated electron (), OH· and H· are produced. Hydroxyl radical is the most effective for producing protein damage, contributing to protein denaturalization, which has been pointed out as the trigger of cell stress [21]. Lymphocytes response is only through Hsp70 expression, we found no evidence of Hsp25, Hsp60, Hsp90 expression when human lymphocytes were irradiated in a low strength gamma ray field. The released Hsp70 protein bounds to aberrant polypeptides/proteins, due to the central role that it plays in the folding and unfolding of other proteins[2, 22].

All sets of lymphocytes shown approximately the same level of Hsp70 expression for 35.2 and 70.5 mGy gamma-ray dose, differences could be attributed to individual response. After 70.5 mGy gamma dose, the relative amount of Hsp70 protein depends upon gamma rays dose. This, makes the Hsp70 protein quantification a possible biodosimeter, sensitive to low intensity gamma-ray radiation field, in the same sense as it is used in biomonitoring and environmental toxicology [23]. However, further and extensive investigations are required before Hsp70 expression is used as biodosimeter. In search for ionizing radiation biodosimeter Benderitter et al. [15] investigated structural membrane modifications in lymphocytes and erythrocytes and Doltchinkonva et al. [14] looked for alterations on electrokinetic properties of purple membranes on Halobacterium halobium cells.

The RW, due his professional activity, has generated certain degree of tolerance to gamma rays (gammatolerance) in comparison with NRWs. This could be the probable explanation to differences on Hsp70 expression observed in lymphocytes after 70.5 mGy gamma dose. This is in agreement with similar situations found in other types of cells and stressors [2, 7, 9, 11, 24-27], and using different ways to measure the adaptive response to ionizing radiation [28]. RW lymphocytes behavior indicates a gamma-ray threshold before human lymphocytes are stressed due to ionizing radiation.