Effect of aluminium on lipid profile and atherogenic index in prepubertal and young adult female rats: A pilot study

INTRODUCTION

Aluminium (Al) is an extensively scattered metal in the environment,^[1] naturally occurring in its trivalent state (Al⁺³) forming organic and inorganic complexes.^[2,3] Even though it is an abundantly present metal, the useful physiological role of Al in the human body is not reported, and its exposure is unsafe.^[4] Excess Al exposure causes toxic effect which perturbs the entire physiology, including alteration of metabolic pathways, like lipid metabolism.^[5] Considering the current worldwide health scenario,^[6] dyslipidaemia and the associated health problems need urgent attention.

Dyslipidaemia is linked with various neurological and cardiovascular diseases.^[7,8] Interestingly, gender has a role in the lipid profile of dyslipidaemic patients.^[9] Before menopause, females have a lower risk of atherosclerotic cardiac disease compared with the age-matched males. Reports indicate that women have a 10-year delay in the first event of myocardial infarction compared to men.^[10,11] Unlike males, different dyslipidaemic indices include reduced low and very-low-density lipoproteins (LDL and VLDL, atherogenic factor) and raised high-density lipoproteins (HDL, atheroprotective factor) in women contribute to a lower risk of cardiovascular disorder in them.^[12]

The steroid hormones, mainly oestrogen have been proposed to contribute to these differences in lipid levels and metabolism in and around puberty.^[13-16] There is also evidence that in women the time of puberty onset leads to altered lipid levels.^[17] Following menopause, a reduction in circulating oestrogen levels increases susceptibility to developing metabolic disorders in women.^[16] Exposure to many environmental and synthetic products (endocrine disruptors) during and before puberty affects pubertal timing and leads to long-term disturbance to the hypothalamo– pituitary–gonadal axis.^[18,19] Oestrogen-like endocrine disruptors cause disturbed metabolic effects by intruding with the synthesis, metabolism and/or binding of natural oestrogens.^[20-22]

Metal ions meddling with oestrogen action have been termed metalloestrogen (ME), some of whose physiological roles are implicit.^[23] MEs enhance the proliferation of cells that are oestrogen-dependent for growth.^[24] The relationship between in utero and peripubertal metal exposure with reproductive hormone levels and sexual progression among adolescent girls in Mexico has been investigated. It was found that various metals, including Al, were associated with delayed pubertal progression in the exposed girls.^[25] Moreover, Al has been shown to differentially affect anxiety-like behaviour in rats of two different age groups when exposed during and before puberty.^[26] The possible association between Al as a leading cause of dyslipidaemia has also been indicated.^[27] However, there is a paucity of literature explaining whether and how an environmental toxin like Al, causes dyslipidaemia in and around puberty. Thus, the present study was conceived to evaluate the Al-induced changes in lipid profile in female rats before and after puberty.

MATERIALS AND METHODS

Methodology

Aluminium chloride was procured from Sigma, India. Other chemicals were of analytical grade. The Al-0 group served as control and was treated with normal saline (i.p.), and the Al-5 and Al-ten groups were exposed to Aluminium chloride (AlCl₃) solution (intraperitoneal i.p.), at doses of 5 and 10 mg Al/kg body weight (BW), respectively. The low dose of 5 mg/ kg BW dose of AlCl₃ solution exposures was selected from the previous studies^[26,28] and the other dose was double that of the already reported dose. Both age groups of rats were divided with the help of Random Allocation Software (Version 1.0, May 2004) into three subgroups (Al-0, Al-5 and Al-10), having six animals in each. All the six groups of rats, Al-0, Al-5 and Al-10 groups of PP and YA females, were administered with either saline or AlCl₃ at the two doses for 2 weeks at the same time of the day. The rats were provided with standard laboratory rat chow and drinking water ad libitum throughout the study. The detailed study design is presented in [Figure 1].

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Figure 1:

The experimental design. PP: Prepubertal, YA: Young adult, Al-0: Control, Al-5: 5 mg/kg Aluminium, Al-10: 10 mg/kg Aluminium, EDTA:Etheylene diamine tetra-acetic acid, n: Number.

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After 2 weeks of treatment, blood samples from overnight fasted rats were collected through the retro-orbital puncture and the rats were sacrificed by cervical dislocation. Blood was collected in ethylenediaminetetraacetic acid-coated tubes and centrifuged at 3000 revolutions per minute (RPM) for 15 min at room temperature. The plasma obtained was used to determine the fasting lipid profile. The blood sample for lipid profile was analysed by spectrophotometric analysis (Beckman Coulter analyser, CA, USA) by cholesterol oxidaseperoxidase method (Agappe Diagnostic Ltd, New Delhi). The same enzymatic analysis along with immunoinhibition assay was employed to estimate HDL (Agappe Diagnostic Ltd, New Delhi) and TG (Agappe Diagnostic Ltd, New Delhi). All the protocol was followed as per the manufacturer’s instruction. Serum LDL and VLDL levels were calculated as per the formula described previously.^[29]

1. LDL = Total Cholesterol - ([TG]/5+HDL)

2. VLDL = TG/5

After weighing the whole liver, approximately 100 g of liver tissue was cut, minced and homogenised in phosphate buffer saline (pH 7.4) using the Teflon homogeniser. The total lipid profiling from the liver sample was also done by spectrophotometric kit following the manufacturer’s protocol as adapted from Folch et al.,^[30] Plasma and liver samples were preserved at −40°C. Biochemical processing of plasma and liver homogenates for lipid profile was carried out within 2 days of sacrifice.

From the estimated biochemical parameters, the atherogenic indices (AI) were also calculated, which are – Castelli risk index-I (CRI-I = TC/HDL-C), Castelli Risk Index-II (CRI-II = LDL-C/ HDL-C), atherogenic coefficient (AC = [TC–HDL-C]/HDL-C) and atherogenic index of plasma (AIP = log [(TG)/HDL-C]).^[31]

RESULTS

Two age groups of female Wistar rats (PP and YA) were exposed to two different doses of AlCl₃ and maintained along with the same age groups of control rats. Although, no mortality was recorded during the experimental period; Al-10 treated rats were relatively less active compared to animals of the other two groups (Al-0 and Al-5). No difference in group-wise food and water consumption was noted (data not shown). The Kruskal-Wallis (KW) test detected a significant difference (P = 0.01) between the BW gain of PP rats (Al-0, Al-5 and Al-10) but not in YA groups (P = 0.1), as shown in [Table 1]. The percentage gain of BW during the experimental period was significantly higher in the Al-5-treated PP rats [Table 1]. Similarly, a higher organosomatic index of the liver was noted in the PP rats treated with Al-5 [Table 1] in comparison to the PP Al-0 group only (P = 0.01) and the PP groups showed a significant (P = 0.03) difference in the KW test.

Comparison of serum lipid profiles

All the groups of YA female rats showed significantly higher levels of plasma TG compared to the PP female rats (χ² = 28.7, P < 0.01) [Figure 2]. The Al-exposed YA female rats demonstrated a significantly higher level of plasma TG compared to their control counterpart (P = 0.01), and the plasma TG of Al-10 was significantly higher than the Al-5 YA rats (P = 0.02) [Figure 2a]. Significantly higher plasma cholesterol levels were seen in both Al-exposed YA rats (χ² = 27.3, P < 0.01). Insignificant alterations in plasma cholesterol levels were observed in both doses of Al-exposed PP female rats [Figure 2b]. In the case of plasma VLDL cholesterol [Figure 2c], the alterations are very similar to that of plasma TG. Statistically significant higher level of plasma VLDL cholesterol in Al-exposed YA females compared to PP female rats in a dose-dependent manner is shown (χ² = 28.7, P < 0.01) in [Figure 2c]. The plasma LDL cholesterol levels in control YA females were lower than that of PP control female rats (P = 0.01) [Figure 2d]. These plasma LDL cholesterol levels in control YA female rats were lower than the Al-exposed rats of the same age group; however, it was statistically significant compared to the Al-5 group (P = 0.01) but insignificant when compared to the Al-10 group.

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Figure 2:

Box plot for plasma (a) triglyceride, (b) total cholesterol, (c) VLDL-Cholesterol, (d) LDL cholesterol and (e) HDL cholesterol contents of prepubertal (n: number, n=6 for Al-5 and Al-10 each) and young adult (n: number, n=6 for Al-5 and Al-10 each) female rats exposed to two doses of aluminium (Al). # indicates a significant difference with the prepubertal rats of the same Al exposure group. @ indicates a significant difference with the Al-0 subgroup of the same age group of rats. $ indicates a significant difference with the Al-5 subgroup of the same age group of rats. LDL: Low-density lipoprotein, VLDL: Very-low-density lipoprotein, HDL: High-density lipoprotein.

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From [Figure 2], it is observed that the plasma lipid parameters were undisturbed by the Al exposure to PP female rats; however, plasma HDL cholesterol levels were significantly elevated in the Al-5 group (P = 0.01) and insignificant in the Al-10 group of PP female rats [Figure 2e]. A similar trend of raised plasma HDL cholesterol was observed in Al-exposed YA female rats, but the differences between the groups were statistically insignificant.

DISCUSSION

The exposure to Al caused a significant increase in BW in the Al-5 group of PP female rats. Contrary to this, no significant change in BW gain was observed in both the Al exposed groups compared to the control YA female rats. Wide variations were noted in these data which did not meet statistical significance. These data suggest that an acute Al exposure for just 2 weeks might affect the normal growth and metabolism. Further to corroborate the finding, and to appreciate the metalloestrogenic property of Al exposure, the two doses of Al exposure raised the organosomatic index of the liver in PP female rats only. There are inconsistent reports of Al-induced changes in BW where the exposure duration varied from 4 weeks to lifelong.^[33,34] The acute exposure to Al was limited to 2 weeks as that was the window period available for the PP exposure group between weaning and adolescence.^[35]

In humans, variable changes in fasting lipid profile during puberty have been documented earlier.^[14] We have observed a post-pubertal increase in plasma TG and VLDL cholesterol levels in the YA female rats. The Al-treated YA female rats had higher plasma lipid parameters compared to the PP control group (Al-0/PP). All the liver lipid parameters were relatively lower in Al-exposed YA female rats in comparison to the age-matched control group (Al-0/YA). However, this was not the type of observation seen in PP female rats. The Al-10/PP female rats had higher liver lipid levels in comparison to their age-matched controls (Al-0/PP). On the other hand, the Al-5 group of animals failed to show any rise in the lipid parameters except HDL cholesterol. In general, the main cardioprotective function of the ‘good cholesterol’ HDL is attributed to its ability to act as both the acceptor of cholesterol from cells and as the cholesterol carrier in the reverse cholesterol transport pathway, including delivery to the liver.^[36-38] This facilitates low plasma cholesterol levels in health. In our study, however, we have witnessed a significant decrease in hepatic lipid levels in both Al-5 and Al-10 exposed YA rats only and not in PP rats compared to control groups. This inverse finding in the liver and plasma lipid levels further supports that Al perturbs the lipid metabolism mainly in the YA rats, indicating that Al might lead to dyslipidaemia due to its metalloestrogenic activity. Remarkably, plasma HDL cholesterol of the Al-5/PP rats was the only parameter that showed a significant increase. Similar to the association of elevated HDL in PP obese children,^[39] the increased gain in BW in the Al-5 group of PP female rats might have some correlation.

Alterations in lipid profile with hypertriglyceridemia in Al-exposed animals were reported^[40,41] that ratified the current observations of raised plasma TG levels in Al-5 and Al-10 groups of YA female rats. However, this present report unfolded that the same level of Al exposure for the same duration failed to replicate similar changes in plasma TG levels of PP female rats. Similarly, PP female rats also demonstrated resistance towards changes in total plasma cholesterol level in response to Al-exposures, while YA female rats were not. Studies suggested that interaction between Al and the surface of HDL causes changes in the physicochemical properties of the lipoprotein monolayer.^[42] Interaction between the negatively charged proteins with the Al³⁺ caused its binding to phosphate groups in the phospholipid monolayer at the HDL surface.^[43] These unusual reactions might be responsible for the observed plasma and liver HDL cholesterol levels in Al-exposed animals.

Obstructions of mitochondrial functions in Al-insulted HepG2 cells indicated a disrupted Tricarboxylic acid cycle (TCA) cycle, limiting the adenosine triphosphate production, in turn, with an increase in lipid production.^[44] Thus, Al might be involved in changes in lipid parameters by both direct and indirect processes, as Al-induced alterations in activities of enzymes involved in the synthesis, translocation, as well as peroxidation, also cannot be ruled out. Further to substantiate the finding of our preliminary report, the enzymatic involvement in lipid metabolism after acute Al exposure in female rats in and around puberty must be explored.

Lipid ratios and AI are used to correlate dyslipidaemia with cardiovascular risks. Increases in CRI-I, CRI-II, AC and AIP were noted in Al-exposed groups of YA female rats in comparison to respective groups of PP female rats. These indicated the role of oestrogen in Al-induced alterations in lipid profile and cardiovascular risk factors. In this context, it is worth noting that Al is a ME^[4,45-47] and its involvement in oestrogen-mediated mechanisms had been suggested for long.^[48,49] Mainly, these studies show how Al interferes with the oestrogen receptor signalling. Moreover, alterations in lipid profile in women with menopause^[50] and its correction with oestrogen therapy^[51] had already suggested the crucial role of oestrogen in lipid metabolism and dyslipidaemia. Experiments with ovariectomised rats also confirmed the role of oestrogen in liver lipid homeostasis.^[52,53]

The present study sheds light on the Al-induced dyslipidaemia in YA female rats. In addition, mostly unperturbed lipid parameters, as well as lipid ratios in PP female rats, indicate the metalloestrogenic property of Al. However, the possible role of Al as ME particularly in the context of lipid homeostasis is not fully understood. Nevertheless, the study evinced the contribution of oestrogen in Al toxicity, even in acute exposure. Future studies should be directed towards unfolding the enzymatic mechanism of the metalloestrogenic property of Al, contributing to dyslipidaemia.

CONCLUSION

Variations in lipid parameters of plasma and liver observed in the current experiment suggest that Al possibly interacts differentially with the lipid metabolism depending on the reproductive age of the female rats. This significant finding is the first of its kind where Al exposure has been studied in female rats of two age groups (PP and YA), to reveal the interaction between Al and the oestrogen axis. In general, distinct oestrogen statuses in virtue of their pubertal state related to lipid metabolism have been documented.^[14,54] The result reported here sheds light on the influence of oestrogen status on the Al-induced alterations in lipid profile parameters and cardiovascular risk factors. This is the first report of acute Al exposure in PP and YA female rats suggesting oestrogen’s involvement in Al-induced dyslipidaemia.