Chronic Contamination with 137 Cesium in Rat: Effect on Liver Cholesterol Metabolism

Abstract

After the Chernobyl nuclear accident, epidemiological studies on human populations living in ¹³⁷Cs-contaminated areas revealed the increase frequencies of thyroid cancer and evoked the apparition of cardiovascular diseases, hormonal effect, liver alteration, and lipid disorder. Actually, it raises a problem of public safety for the populations living on these territories that are exposed to low levels of ¹³⁷Cs during a long period through food. Then it is necessary to study potential effect of this chronic contamination. To mimic this situation, the authors investigate the potential biological effects of chronic exposure to ¹³⁷Cs at a postaccidental dose (150 Bq/rat/day) on hepatic metabolism of cholesterol in rat. Plasma lipid level, gene expression and activity were analyzed. It was observed that in ¹³⁷Cs-exposed rats, gene expression of low-density lipoprotein receptor (LDLr), apolipoprotein B (apoB), and liver X receptor α (LXRα) are increased (95%, p < .05; 34%, p < .05; 20%, p < 0.05, respectively), whereas transporter adenosine triphosphate–binding cassette transporter G5 (ABCG5) is decreased (42%, p < .05). In addition, cytochrome P450 27A1 (CYP27A1) activity is increased (34%, p < .05) in contaminated rat liver. In conclusion, the results suggest that ¹³⁷Cs contamination at low-level induces molecular modifications of the liver cholesterol metabolism without leading to a dysregulation of its homeostasis. These results suggest that chronic long term exposure at low-level of ¹³⁷Cs may evolve to lipid disorder.

After the Chernobyl nuclear reactor accident (April 26, 1986), some studies have shown an increased frequency of thyroid diseases (Likhtarov et al. 2005) for people living on the contaminated territories. Several other human pathologies such as cardiovascular diseases (Bandazhevskaya et al. 2004) and alteration of the central nervous system (Ivanov et al. 2000), digestive tract (Dehtiar’ova and Kozlova 2001), liver (Shkala 1998), and gonad (Cheburakov, Cheburakov, and Belozerov 2004) have been evoked on these human populations. In all these works, the radionuclide involved in long term is mainly cesium 137 (¹³⁷Cs).1 Studies of ¹³⁷Cs-exposed animals showed alterations of the hematopoietic system (Norris, Poole, and Rehfeld 1966), appearance of thyroid cancer, or bone cancer (Fritz 1972). The Chernobyl accident is a unique situation that raises a problem of public safety for the populations living in the contaminated areas that are exposed to low levels of ¹³⁷Cs during a long period. The knowledge on ¹³⁷Cs effect concern only cancer effect as all ionizing radiation because of the high dose used. Actually, chronic contamination with low level of ¹³⁷Cs are poorly documented and to our knowledge, animal studies have not been investigated.

In our institute (IRSN), Envirhom program specifically analyses biological effect of radionuclides such as uranium and ¹³⁷Cs. This program started with the analysis of acute and chronic effect of uranium contamination on central nervous system (Lestaevel et al. 2005; Bussy et al. 2006), physiological systems such as hepatic and renal metabolism of xenobiotics (Souidi et al. 2005), cholesterol (Gueguen et al. 2006), and vitamin D (Tissandie et al. 2006b). To proceed with this program and for the 20th anniversary of Chernobyl accident, we are interested in ¹³⁷Cs chronic contamination effect on vitamin D and cholesterol metabolism. Recently, vitamin D metabolism have shown to be affected after 3 months of contamination by ¹³⁷Cs in the rat (Tissandie et al. 2006a). In the same way, and to continue the investigation in this field, we studied in this work the liver cholesterol metabolism. Indeed, altered plasma cholesterol and triglycerides levels have been observed in people exposed to radiation during the Chernobyl accident (Chaialo et al. 1991). It is actually accepted that liver cholesterol metabolism is primordial for its plasmatic homeostasis (Ory 2004) and its disruption causes liver and cardiovascular diseases such as atherosclerosis and dyslipidemia (Libby, Aikawa, and Schonbeck 2000). Thus, some parameters involved in liver cholesterol metabolism such as enzymes (CYP7A1, CYP27A1, HMGCoAR, and HMG-CoAS), lipoprotein receptors (LDLr and SR-BI), sterol transporters (ABCA1 and ABCG5), apolipoproteins (ApoA1, ApoB, and ApoE), and nuclear receptors involved in liver cholesterol metabolism regulation (FXR, LXRα, PPARα, and RXR) were analyzed.

The objectives of this work were thus to investigate the potential biological effects of chronic exposure to ¹³⁷Cs at a postaccidental dose (150 Bq/rat/day) (Handl et al. 2003) on hepatic metabolism of cholesterol in rat. For the first time, we studied, on an animal model the plasma and liver cholesterol parameters, hepatic gene expression of lipoprotein receptors, sterol transporters, apolipoproteins, enzymes, and nuclear receptors.

MATERIALS AND METHODS

Animals

Sprague-Dawley male rats (Charles River, France), weighing 250 g each, were divided into two groups of 10 rats each: a control group and an experimental group. All experimental procedures were approved by the Animal Care Committee of the Institute of Radioprotection and Nuclear Safety and complied with French regulations for animal experimentation (Ministry of Agriculture Act number 87–848, October 19, 1987, modified May 29, 2001).

Contamination

The rats in the experimental group were exposed to ¹³⁷Cs in their drinking water for 3 months, at a dose of 6500 Bq/L (150 Bq/rat/day) (CERCA, Pierrelatte, France).

Clinical and Biochemical Parameters Assays

We used an automated Konelab 20 to measure plasma ALT, AST, GGT, AP, bilirubin, albumine, total protein, total cholesterol, LDL-C, HDL-C, phospholipids, and triglycerides (biological chemistry reagents, Thermo Electron Corporation, France) in the control and ¹³⁷Cs-exposed rats.

Cholesterol concentration in liver samples were measured using technique previously described (Boehler et al. 1999).

Preparation of Liver Microsomes and Mitochondria

Liver microsomes and mitochondria were prepared from fresh samples (about 1 g) according to (Souidi et al. 1999).

Determination of CYP7A1 and CYP27A1 Enzyme Activities in the Liver

The radio isotopic assays for microsomal CYP7A1 and mitochondrial CYP27A1 (Souidi et al. 1999) activities in the liver have been described in detail previously.

Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)

Real-time PCR was used to analyze the mRNA expression of the CYP7A1, CYP27A1, HMGCoAR, HMGCoAS, LDLr, SR-BI, ABCA1, ABCG5, ApoA1, ApoB, ApoE, RXR, FXR, LXRα, and PPARα.

Total RNA was prepared with the RNeasy total RNA isolation Kit (Qiagen, France) according to the manufacturer’s instructions. The cDNA was produced from 1 μg of total RNA by reverse transcription with BD Sprint PowerScipt PrePrimed 96 Plate (BD Biosciences Clontech, France). PCR amplification used Syber PCR master mix (PE Applied). Optimized PCR used the Abi Prism 7000 Sequence detection system (Applied Biosystems). Samples values were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) values and fold-changes calculated to the control group.

Statistical Analysis

Results are reported as means ± SE. Statistical analyses were performed with Student’s t test. Differences were considered significant when p < .05.

RESULTS

Table 1 shows the physical and plasma biochemical data for rats contaminated with ¹³⁷Cs for 3 months (150 Bq/rat/day) and control rats. The experimental contamination of this study did not affect food intake, weight gain, or the animals’ general health status. Concentrations of biochemical markers associated with liver function (ALT, AST) were unaffected. Plasma concentration of cholesterol, phospholipids, and triglycerides were similar between the control and ¹³⁷Cs-exposed animals. In the liver, cholesterol ester and free cholesterol were unaffected in ¹³⁷Cs-exposed animals compared to control.

The effects of this 3-month contamination on the liver gene expression of enzymes (CYP7A1, CYP27A1, HMG-CoAR, HMGCoAS), nuclear receptors (FXR, LXRα, PPARα, RXR), lipoprotein receptors (LDLr, SR-BI), sterol transporters (ABCA1, ABCG5), and apolipoproteins (ApoA1, ApoB, ApoE) in rats are reported in Figure 1. The mRNA level of the reference gene (HPRT) was unchanged in the liver after 3 months of contamination with ¹³⁷Cs. LDLr mRNA level increased in ¹³⁷Cs exposed rats (95%, p < .05). Similarly, the ApoB mRNA level increased (34%, p < .05) but ABCG5 mRNA expression decreased (42%, p < .05). We evaluated the effects of ¹³⁷Cs contamination on the gene expression of the nuclear receptors LXRα, PPARα, and RXR. As Figure 1 shows, LXRα mRNA level increased in the liver (20%, p < .05) compared with controls. ¹³⁷Cs contamination did not change gene expression of PPARα and RXR.

Hepatic CYP7A1 and CYP27A1 enzymatic activities were evaluated in the control and contaminated rats (Figure 2). The CYP27A1 activity increased by 34% (p < .05) compared to controls. CYP7A1 activity was not modified by ¹³⁷Cs contamination.

DISCUSSION

After the Chernobyl nuclear accident and scattering of radionuclides like ¹³⁷Cs in the environment, many people were exposed until today due to the physical long half-life of ¹³⁷Cs (30 years). Epidemiological studies on human populations living on ¹³⁷Cs-contaminated areas revealed the increased frequency of several diseases particularly for highly exposed people: thyroid cancer (Cherenko et al. 2004) cardiovascular symptom in children (Bandazhevskaya et al. 2004), and hormonal effect (Goncharov et al. 1998). The rare studies done on animals essentially showed effect of ¹³⁷Cs on haematopoietic system (Norris, Poole, and Rehfeld 1966) and some cancers (Nikula et al. 1996). Indeed, it raises a problem of public safety for the populations living on these territories that are exposed to low levels of ¹³⁷Cs during a long period through food. Then it is necessary to study potential effect of this chronic contamination. To examine the effect of ¹³⁷Cs chronic contamination with environmental concentration (contaminated territories), recently, in our laboratory, in vivo effect of radionuclides were studied on various physiological systems. In this, central nervous modifications at electrophysiological level (Lestaevel et al. 2006) and vitamin D metabolism disturbance were observed after chronic exposure to ¹³⁷Cs (Tissandie et al. 2006b). In the same way, the effect of ¹³⁷Cs chronic contamination on liver cholesterol metabolism was studied. In some studies altered plasma cholesterol and triglycerides levels have been observed in people exposed to radiation during the Chernobyl accident (Chaialo et al. 1991). Theses results suggested that liver cholesterol metabolism was a target of ¹³⁷Cs. In order to mimic Chernobyl environmental exposure, we have studied ¹³⁷Cs effect on rat contaminated through drinking water (6500 Bq/L) at a dose corresponding to the maximum concentration measured in food chain in Belarus immediately after the Chernobyl accident (Handl et al. 2003). The rats were exposed to low level of ¹³⁷Cs by chronic ingestion for 3 months that didn’t affect their food intake, weight, or general health status. Blood tests showed no effect on hepatic function markers such as ALT and AST, confirming that the dose used in this study was not hepatotoxic. Nevertheless, toxicity experience using ¹³⁷Cs single administration showed elevation of plasma transaminases level (Stojadinovic and Jovanovic 1966), suggesting that the liver could be a biological target of ¹³⁷Cs. Highly exposed people to ionizing radiation during Chernobyl accident (liquidators) have also altered liver histology (Liubchenko et al. 1994). Similar impairments were observed in rats living within the accident zone (Pinchuk et al. 1991). In our conditions of low-dose contamination, we didn’t observed macroscopic alteration of the liver.

In addition, people exposed to radiation during the Chernobyl accident have altered plasma cholesterol and triglycerides, levels that could lead to cardiovascular diseases (Chaialo et al. 1991). In our study, the plasma level of cholesterol, triglycerides, and phospholipids were not affected after a 3-month contamination at a dose of 150 Bq/rat/day. These discrepancies are probably due to the dose of ¹³⁷Cs used and the duration of the contamination in our experimental condition. On the other hand, we have observed molecular modifications in the liver of ¹³⁷Cs-exposed rats. Indeed, we observed that in ¹³⁷Cs-exposed rats, gene expression of LDLr responsible of hepatic capture of circulating cholesterol was increased. In parallel, we observed an increased mRNA level of apoB, a LDLr ligand. The up-regulation of LDLr and apoB in the liver suggests that hepatic capture of cholesterol was changed. Moreover, the transporter ABCG5, responsible for sterol elimination and more precisely for elimination of cholesterol in the bile, was decreased in contaminated rat. In contrast, CYP27A1 activity, a key enzyme of cholesterol transformation in bile acid, was increased. Some studies observed that high ionizing radiations lead to modifications of cholesterol metabolism (Feurgard et al. 1999; Scanff et al. 2004). Surprisingly, we observed molecular modification of gene involved in cholesterol capture, synthesis, and degradation with environmental low dose of ¹³⁷Cs. This dysregulation of cholesterol metabolism was associated with a modification LXRα gene expression, a crucial nuclear receptor for the control of lipid homeostasis (Ulven et al. 2005), without leading to its alteration. This could be explained by a chronic contamination too short. In conclusion, these results indicate that the chronic exposure with low level of ¹³⁷Cs lead to subtle molecular modifications without indication of toxicity. This study suggests that long-term chronic contamination could evolve to lipid disorder.