NF-κB regulates the response of embryonic cells to heat shock

Abstract

NF-κB was shown previously to regulate apoptotic cell death processes in various experimental systems. However, its role in controlling teratogen-induced cell death has not been established yet. Therefore, the objective of the present study was to explore the involvement of the p65 subunit of NF-κB in the response of mouse embryonic fibroblasts (MEFs) to heat shock, using p65 knockout (p65^−/−) cells. Indeed, we found p65^−/− MEFs to be more susceptible to the exposure to heat shock, as compared with wild-type (WT) MEFs, as they demonstrated a more prominent decrease in cell survival and proliferation as well as the appearance of cells undergoing apoptotic cell death. These heat-shock-induced effects were preceded by a decrease in p65 expression in WT cells, which was accompanied by a decrease in IκBα expression in WT MEFs, while disappearing completely in p65^−/− MEFs and accordingly, by an increase in p-IκBα expression in both cell lines, which was found to be more prominent in p65^−/− MEFs. Interestingly, the heat shock-induced decrease in p65 expression was accompanied by an increase in HSP70 expression in both cell lines. However, it was again found to be more prominent in p65^−/− MEFs. Taken together, our results suggest a protective role for the p65 subunit of NF-κB in mechanisms underlying the response of embryonic cells to heat shock.

• Heat shock
• apoptosis
• MEFs
• p65
• NF-κB
• embryo

Introduction

Maternal hyperthermia was shown to be a potent teratogen in experimental animals as well as in humans [1–3], causing mainly embryonic head and brain anomalies [4]. More than that, anomaly formation was shown to be preceded by an apoptotic process in target embryonic organs [5], [6] as well as by induction of the expression of several heat shock proteins (HSPs) [7], [8]. Thus, we have shown in our previous study that the heat-shock-induced teratogenic effect was accompanied by a time-dependent appearance of apoptotic cells in the affected embryos as well as by an increased level of HSP60 [9]. One of the central genes involved in the regulation of the apoptotic process is the transcription factor NF-κB, which was shown to function as a pro- or an anti-apoptotic molecule, depending most probably on the type of apoptotic trigger or cells involved [10–13]. Thus, experiments performed in our laboratory demonstrated an anti-apoptotic role for NF-κB, when they revealed that the teratogenic activity of cyclophosphamide (CP), which is associated with the appearance of apoptotic cells in embryonic target organs, is also accompanied by suppression of NF-κB DNA-binding activity in the embryonic brain, which is known as a target organ for CP [14]. A decrease in NF-κB DNA-binding activity was detected also following exposure to thalidomide [15] or ethanol [16]. On the other hand, Molestina et al. showed that infection of mouse embryonic fibroblasts with Toxoplasma gondii resulted in NF-κB activation and increased expression of anti-apoptotic genes [17].

An interesting correlation was found between the cellular response to heat shock and NF-κB activation by other stress conditions. Thus, several studies have shown that exposure of animals to heat shock prior to exposure to tumor necrosis factor (TNF) α or ionizing irradiation or induction of gastrointestinal inflammation can inhibit NF-κB activation, which was induced by those stress conditions [18–22]. More than that, this heat-shock-induced decrease in NF-κB activation was accompanied by an increase in the expression of various HSPs [20]. Also, Guzhova et al. showed that exposure of cells to heat shock resulted in formation of complexes of HSP70 with various subunits of NF-κB, among them p65 [23]. However, this finding is still controversial, since Malhotra et al. showed that exposure to heat shock can inhibit NF-κB activation in the absence of HSPs expression [24]. Altogether, the above data suggest a possible interaction between NF-κB and HSPs as part of the cellular response to heat shock; however, the exact mechanisms underlying this interaction are not fully understood. Therefore, in the present study we tried to analyze further the involvement of NF-κB in the embryonic response to heat shock. The most suitable experimental model for such a research would be p65 knockout embryos. However, since those embryos do not survive to term [25], this goal was achieved by using an in vitro model employing mouse embryonic fibroblasts (MEFs) derived from such p65 knockout (p65^−/−) or wild-type (WT) embryos. The response of both cell lines to heat shock was analyzed by following up several parameters, such as cell survival and proliferation, cell death and changes in the expression of molecules associated with the cellular response to heat shock, such as p65 and its inhibitory protein IκBα as well as HSP70.

Materials and methods

Cell culture

Mouse embryonic fibroblasts (MEFs) derived from p65 knockout (p65^−/−) or wild-type (WT) embryos were kindly provided by A. Hoffmann (University of California, San Diego, CA, USA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn calf serum (NBCS), 2 mM L-Glutamine, 100 U/ml Penicillin, 100 µg/ml Streptomycin and 12.5 U/ml Nystatin (Biological industries, Beith Haemek, Israel) in a humidified incubator (37°C, 10% CO₂).

Exposure to heat shock and determination of cell survival

MEFs were exposed to heat shock 24 h after plating (2–6 × 10⁵ cells/35- or 60-mm tissue culture plates, respectively). The plates were placed in a plastic bag, which was filled with a mixture of 10% CO₂ and 90% air before sealing and subjected to a 1-h heat shock at 45°C in a dry incubator. Cells were allowed to recover at 37°C for various periods of time (24–72 h), harvested and stained with trypan blue to determine cell survival and subjected to the assays listed below.

Cell proliferation assay

Evaluation of changes in cell proliferation following exposure to heat shock was performed by measuring 5′-bromo-2′-deoxyuridine (BrdU) (Sigma, Rehovot, Israel) incorporation. Cells were harvested following exposure to 50 µM BrdU for 4 h, washed in phosphate-buffered saline (PBS) and resuspended in NBCS before fixation in 70% ethanol overnight at 4°C. Following two washes in washing solution (PBS containing 0.1% Bovine serum albumin (BSA), 0.01% sodium azide and 0.05% Triton-X 100), DNA was denatured by 1 N HCl for 5 min and excess of acid was neutralized by 0.1 M sodium borate, pH 8.5, for 10 min. Then, cells were washed twice in washing solution and incubated for 1 h with mouse anti-mouse BrdU (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 2 µg/ml), rinsed in washing solution and incubated for 30 min with Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA; Fab fragments; 30 µg/ml). Following two washes in washing solution, cells were stained with 50 µg/ml propidium iodide (PI; Sigma, Rehovot, Israel). Ten thousand cells/sample were analyzed by a Fluorescence activated cell sorter (FACS; FacSort, Becton Dickinson FACS system, Mountain View, CA) and the percentage of cells in the S-stage of the cell cycle was evaluated by the WinMDI 2.1 software.

Cell cycle analysis

Nuclei from all experimental groups were isolated by resuspending the cells in a hypotonic fluorochrome solution containing 50 µg/ml PI, 0.1% sodium citrate and 0.1% Triton X-100. Ten thousand nuclei/sample were analyzed by FACS and the percentage of cells in the various stages of the cell cycle was evaluated by the WinMDI 2.1 software.

Confocal microscopy

Cells from the various experimental groups grown on coverslips were rinsed twice with PBS, fixed in methanol for 5 min at −20°C and rinsed twice with a washing solution composed of PBS supplemented by 0.1% bovine serum albumin (BSA) and 0.1% sodium azide. Cells were incubated with primary antibodies against p65, IκBα or p-IκBα (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or HSP70 (Stressgene Biotechnologies, Victoria, BC, Canada), diluted in the washing solution at 0.4–2 µg/ml, for 1 h at 4°C. Following two washes in the washing solution, the primary antibody binding was visualized by incubation with FITC-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, USA; 2 µg/ml), diluted in the washing solution, for 30 min at 4°C. Following another wash, coverslips were stained with PI (50 µg/ml) diluted in PBS, mounted onto slides and examined under an LSM 410 Inverted Laser Scan Microscope (Zeiss, Germany).

Western blot analysis

Cells from the various experimental groups were lysed in ice-cold RIPA buffer, containing 1% NP40, 0.1% SDS, 0.5% Sodium Deoxycholate and 14.3% protease inhibitors (Complete, Roche Diagnostics, Mannheim, Germany) in PBS and centrifuged for 10 min at 14 000 rpm at 4°C. Protein concentration was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples containing 50–100 µg protein were resolved by electrophoresis on a 12% SDS-polyacrylamide gel and a Prestained M.W. standard (Precision Plus Protein Standard, Bio Rad Laboratories, Hercules, CA, USA) was used as a marker. Proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) and nonspecific binding sites on the blots were blocked by incubation in 5% low-fat dried milk in buffer containing 0.15 M NaCl, 0.02 M Tris Base pH 7.4 and 0.1% Tween 20 in DDW (TBS-Tween) for 2 h at room temperature. Proteins were detected by incubation overnight at 4°C with primary antibodies against p65, IκBα, p-IκBα or Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or HSP70 (Stressgen Biotechnologies, Victoria, BC, Canada) (0.2–1 µg/ml). Membranes were washed twice with TBS-Tween and incubated with Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in TBS-Tween at 0.1–0.2 µg/ml for 45 min at room temperature. After washing twice in TBS-Tween, the membranes were incubated with ECL reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and exposed to X-ray film. Quantitation of the data was performed by densitometry using a Renium 202D Bio Imaging System and Tina PC Bas software.

Statistical analysis

Statistical analysis of the survival rate, the percentage of dead cells and cell cycle changes was performed by Student's t-test and the level of significant was p < 0.05.

Results

Effect of heat shock on cell survival

Heat-shock-induced changes in cell survival were evaluated at different time points after exposure by counting trypan-blue-stained cells (Figure 1). Both cell lines exhibited a significant decrease in cell survival, which became visible at 48 h after exposure. However, 72 h after exposure to heat shock, a clear difference in the susceptibility of both cell lines to heat shock was noted, when p65^−/− MEFs exhibited a more profound decrease in cell survival, as compared with WT cells (Figure 1a, b). Accordingly, the percentage of dead (trypan-blue-stained) cells was found to be significantly higher among p65^−/− as compared with WT MEFs, when tested at 72 h after exposure to heat shock (Figure 1c).

Figure 1. Heat-shock-induced changes in cell survival of WT (a) or p65^−/− (b) MEFs at various time points after exposure. (c) The percentage of trypan blue-positive cells, tested at 72 h after exposure to heat shock. Data are presented as mean of eight experiments ± SD.

Heat-shock-induced changes in cell proliferation

Heat-shock-induced changes in cell proliferation were examined by flow cytometric analysis of BrdU incorporation by both cell lines, 24 h after exposure. As can be seen in Figure 2, both cell lines exhibited a decreased proliferation rate; however, it was found to be more prominent in p65^−/− (Figure 2c, d), as compared with WT MEFs (Figure 2a, b), suggesting an higher sensitivity of this cell line to the teratogen.

Figure 2. Flow cytometric analysis of BrdU incorporation by WT (a, b) or p65^−/− (c, d) MEFs that were not treated (a, c) or heat shocked and tested 24 h later (b, d). The gated cells represent the percentage of WT or p65^−/− MEFs in the S-phase of the cell cycle (1 representative out of 4 experiments).

Cell cycle analysis of heat-shock-treated MEFs

Time-dependent changes in the percentage of cells in the various stages of the cell cycle following exposure to heat shock were examined by flow cytometry. Indeed, both cell lines differed in their response to the exposure to heat shock, demonstrating changes primarily in the Sub-G₁ or S phases of the cell cycle, representing cells undergoing apoptotic cell death or S arrest, respectively. Thus, 24 or 48 h after exposure to heat shock, no clear changes in the proportion of cells of both cell lines in the Sub-G₁ phase of the cell cycle were observed (Figure 3a, b). However, 72 h after treatment, a significant increase in the proportion of p65^−/− MEFs in the Sub-G₁ phase of the cell cycle was detected, while no such change was exhibited by WT cells (Figure 3a, b, e). Similarly, exposure to heat shock demonstrated no clear changes in the percentage of WT cells in the S phase of the cell cycle at all time points tested (Figure 3c, e), while FACS analysis of p65^−/− cells revealed an heat shock-induced S arrest, that became significant at 48 and 72 h after exposure (Figure 3d, e).

Figure 3. Heat-shock-induced time-dependent changes in the percentage of WT (a, c) or p65^−/− (b, d) cells in the Sub-G₁ (a, b) or S (c, d) phases of the cell cycle (mean of 8 experiments ± SD). Flow cytometry histograms, representing cell cycle changes in WT or p65^−/− MEFs 72 h after exposure to heat shock, are shown in (e). Sub-G₁, G₀\G₁, S, G₂\M-phases of the cell cycle.

Heat-shock-induced changes in the expression and intracellular localization of heat-shock-associated molecules

Possible changes in whole cell expression and intracellular localization of p65, IκBα p-IκBα and HSP70 in both cell lines following exposure to heat shock were examined by western blotting and confocal microscopy.

p65

As can be seen in Figure 4, western blot analysis revealed a decrease in p65 whole cell expression by WT MEFs following exposure to heat shock, which was found to be most prominent 4 h after exposure (Figure 4a). These results were supported by confocal microscopy analysis, demonstrating some heat-shock-induced decrease in p65 expression in the cytoplasm as well as its translocation to the nucleus and accumulation at the nuclear periphery (Figure 5a, b).

Figure 4. Western blot analysis of the expression of p65 (a), actin (b) or HSP70 (c, d) by WT (a, b, c) or p65^−/− (d) MEFs, at various time points after exposure to heat shock (1 representative out of 3 experiments).

Figure 5. Confocal microscopy analysis of the intracellular localization of p65 (a, b) or IκBα (c–f) in WT (a–d) or p65^−/− (e, f) cells that were not exposed (a, c, e) or exposed to heat shock and analyzed 4 h later (b, d, f) (red staining: nuclei stained with PI; green staining: expression of p65 or IκBα) (1 representative out of 3 experiments) (X450).

IκBα

Similarly to the data obtained for p65, western blot analysis demonstrated a decrease in IκBα whole cell expression by WT MEFs following exposure to heat shock, which was found to be most profound 4 h after the exposure (Figure 6a). Interestingly, no IκBα expression was detected in heat-shocked p65^−/− MEFs at all time points tested (Figure 6b). These results were supported by confocal microscopy analysis, when 4 h after exposure to heat shock some decrease in the cytoplasmic expression of IκBα was demonstrated in WT MEFs (Figure 5c, d), while no expression of the protein was detected in the nuclei of p65^−/− MEFs (Figure 5e, f).

Figure 6. Western blot analysis of the expression of IκBα (a, b) or p-IκBα (c, d) by WT (a, c) or p65^−/− (b, d) MEFs, at various time points after exposure to heat shock (1 representative out of 3 experiments).

p-IκBα

In accordance with the heat-shock-induced decrease or disappearance of IκBα expression in WT or p65^−/− MEFs, respectively, western blot analysis revealed a heat-shock-induced, time-dependent increase in the expression of the phosphorylated form of the protein, which started to be evident 4 h after exposure; however, it was found to be more prominent in p65^−/− MEFs (Figure 6c, d). Interestingly, non-treated cells from both cell lines demonstrated no expression of p-IκBα (Figure 6c, d).

HSP70

Western blot analysis demonstrated a heat-shock-induced, time-dependent increase in the expression of HSP70 in both cell lines, which was recorded first at 4 h after exposure; however, it was again found to be more prominent in p65^−/− MEFs (Figure 4c, d). In agreement with these results, confocal microscopy analysis revealed also a time-dependent increase in HSP70 expression in the nucleus and cytoplasm of cells from both cell lines, which was also visible first at 4 h after exposure (Figure 7b, e). However, a difference in HSP70 expression between the two cell lines was observed 24 h after exposure, when most WT MEFs demonstrated a normal morphology and a high expression of the protein, while in p65^−/− MEFs only part of the cells expressed the protein and those negative for its expression exhibited morphological changes characteristic of different stages of apoptosis (Figure 7c and 7f, respectively). Interestingly, western blot analysis demonstrated no expression of HSP70 in non-treated WT MEFs, while p65^−/− MEFs exhibited a low expression of the protein at either time point tested (Figure c, d). These results were supported by confocal microscopy analysis, demonstrating a low expression of the protein in non-treated p65^−/− but not WT MEFs, localized mainly to the cytoplasm (Figure 7a, d).

Figure 7. Confocal microscopy analysis of the intracellular localization of HSP70 in WT (a–c) or p65^−/− (d–f) cells that were not exposed (a, d) or exposed to heat shock and analyzed at 4 h (b, e) or 24 h (c, f) later (red staining: nuclei stained with PI; green staining: expression of HSP70) (1 representative out of 3 experiments) (X450).

Discussion

The involvement of NF-κB in mechanisms underlying teratogen-induced apoptotic cell death is far from being understood. Thus, in our present study we used p65^−/− MEFs that were exposed to heat shock in order to explore the involvement of the p65 subunit of NF-κB in the response of these cells to the teratogen.

Indeed, studies performed in various cellular systems, demonstrated the ability of heat shock to decrease cell survival rate [26–29]. In agreement with those data, our results demonstrated as well that exposure of MEFs to heat shock resulted in a time-dependent decrease in cell survival. However, this effect was found to be more prominent in p65^−/− compared with WT MEFs and was accompanied by a higher percentage of dead cells, suggesting a protective role for p65 against the hazardous effect of exposure to heat shock. Similarly, Beg and Baltimore demonstrated that the survival rate of WT MEFs exposed to TNFα was higher than that of p65^−/− MEFs [13]. Also, Wang et al. have shown that inhibition of NF-κB activity by an IκBα mutant resulted in a decrease in cell survival [30], while in the study by Molestina et al. activation of NF-κB was accompanied by an increased resistance of WT but not p65^−/− MEFs to apoptotic cell death [17].

The heat-shock-induced decrease in cell survival might be attributed either to a decrease in cell proliferation or to an increase in apoptotic cell death or to the combination of both. Indeed, a heat-shock-induced decrease in proliferation rate was demonstrated in several experimental systems, such as human fibroblasts, vascular smooth muscle, melanoma or malignant glioma cells [28], [29], [31], [32]. In agreement with those studies, our data showed as well that exposure of MEFs to heat shock resulted in a clear decrease in the percentage of cells that incorporated BrdU, which was again more prominent in p65^−/− cells. Those data were supported by cell cycle arrest, characterized by an accumulation of p65^−/− but not WT cells in the S-phase of the cell cycle. Altogether, these results suggest that the lower survival rate of p65^−/− cells in response to heat shock might be caused, at least partially, by a decrease in cell proliferation, which might be attributed to the lack of the p65 protein. Similarly, Autieri et al. blocked the activity of p65 in human vascular smooth muscle cells by antisense oligonucleotides and demonstrated a significant decrease in cell proliferation [33]. Also, Bharti et al. demonstrated that down regulation of NF-κB leads to the suppression of proliferation and arrest of cells between the G₁- and S-phases of the cell cycle [34].

In agreement with the more prominent heat-shock-induced decrease in cell survival and proliferation, p65^−/− cells also exhibited a high percentage of nuclei in the Sub-G1-phase of the cell cycle, suggesting the appearance of cells undergoing apoptotic cell death, which was not detected in WT cells. Our data are supported by the study of Li et al., demonstrating the ability of double-stranded RNA to induce apoptosis in p65^−/− but not in WT MEFs [35], while Starenki et al. showed that inhibition of NF-κB activity resulted in increased cell death following exposure to ionizing irradiation [36]. Also, as mentioned earlier, Bharti et al. demonstrated that down-regulation of NF-κB activity in myeloma cells resulted in induction of apoptosis [34]. Altogether, these results suggest that the absence of p65 might increase the sensitivity of p65^−/− cells to heat shock and implicate this molecule to be involved in mechanisms that protect cells from undergoing apoptosis. However, the possibility that cell death mechanisms other than apoptosis are triggered by exposure to heat shock cannot be excluded [37].

The suggested anti-apoptotic activity of p65 was further supported by the heat-shock-induced decrease in p65 expression detected in WT MEFs, which was found to be most prominent 4 h after exposure and continued to a lesser extent 24 h later. Similarly, studies performed by us and by others demonstrated that exposure to other teratogens, such as cyclophosphamide, thalidomide or ethanol, resulted in suppression of NF-κB DNA-binding activity in several experimental systems [14–16]. At the same time, confocal microscopy analysis demonstrated some heat-shock-induced decrease in p65 expression in the cytoplasm as well as its translocation to the nucleus and accumulation at the nuclear periphery. This movement towards the nucleus might represent a new cycle of activation of the molecule, which might be initiated while the previous cycle is not as yet completed. Similarly, other studies demonstrated a translocation of p65 into the nucleus following exposure to heat shock or to the parasite Toxoplasma gondii[17], [38], [39].

A pattern of expression similar to that of p65 was observed for IκBα, demonstrating a decrease in its expression in WT MEFs following exposure to heat shock, while no IκBα expression was detected in heat-shocked p65^−/− MEFs at either time-point tested. This result might emphasize the importance of the interaction between IκBα and p65, compared with its interaction with other subunits of NF-κB, as part of the cellular response to heat shock. Our data are supported by those of Hoffmann et al., demonstrating a disappearance of IκBα expression in p65^−/− MEFs in response to TNFα, as opposed to WT cells, in which NF-κB activation is accompanied by several quick degradation and synthesis cycles of IκBα [40]. Accordingly, western blot analysis revealed a heat-shock-induced, time-dependent increase in the expression of p-IκBα, which was found to be more prominent in p65^−/− MEFs. Based on these data, we can hypothesize that the decrease in IκBα expression and the increase in its phosphorylated form represent a putative mechanism that enables the transfer of p65 into the nucleus. Accordingly, a decrease in IκBα expression was shown also in other experimental systems, such as vascular endothelial or thyroid cancer cells, which were exposed to TNFα or to ionizing irradiation, respectively [36], [41]. The fact that no IκBα expression was detected in heat-shocked p65^−/− MEFs, accompanied by a more prominent increase in p-IκBα expression in those cells, might suggest an interaction of IκBα with other subunits of NF-κB as part of the cellular response to heat shock. Indeed, this assumption is supported by studies demonstrating an interaction between IκBα and different subunits of NF-κB other than p65, such as p50, p52 or c-Rel [42], [43]. More than that, since IκBα is expressed in both non-treated WT and p65^−/− MEFs, it can be assumed that such an interaction exists also in non-treated p65^−/− cells and is augmented upon exposure to heat shock.

Based on studies addressing the involvement of the NF-κB family in the cellular response to heat shock[18], [44], [45], we tried to establish a possible correlation between p65 and HSP70 expression in heat-shocked MEFs. Indeed, western blot analysis demonstrated a heat-shock-induced, time-dependent increase in the expression of HSP70 in both cell lines, detected first 4 h after the exposure, which was found to be more prominent in p65^−/− MEFs. Similarly, an increase in HSP70 expression was detected in various experimental systems following exposure to heat shock, which was shown to protect the cells from apoptosis and, thus, to promote cell survival [46], [47]. In agreement with the above studies, the more prominent increase in the expression of HSP70 detected in p65^−/− MEFs might represent an alternative mechanism aimed at protecting the cells against the exposure to heat shock, which is activated in the absence of p65 and thus might be responsible, at least partially, for the survival of those cells. This assumption is supported by confocal microscopy analysis, demonstrating a normal morphology and a high expression of HSP70 in most WT MEFs 24 h after exposure to heat shock, while many p65^−/− MEFs exhibited morphological changes characteristic of an apoptotic process and almost no expression of the protein. More than that, non-treated p65^−/− MEFs were found to express a low level of HSP70 at all time points tested, while it was absent completely in WT cells, suggesting the existence of a HSP70-mediated protective mechanism in non-treated p65^−/− cells as well.

Taken together, p65^−/− MEFs were found by us to be more sensitive to exposure to heat shock, demonstrated by a more prominent effect of the teratogen on cell survival and proliferation, cell cycle arrest and induction of apoptotic cell death as well as changes in the expression of the apoptosis-regulating molecules p65, IκBα and HSP70, which differed between the two cell lines. Our results suggest a protective role for the p65 subunit of NF-κB in the response of embryonic cells to heat shock.

Acknowledgements

This work was supported by grant No. 6234-1 from The Israel Ministry of Health.