Biochemical profiling of metabolomics in heavy metal-intoxicated impaired metabolism and its amelioration using plant-based bioactive compound

Abstract

Exposure to Pb is widely spreading and has far-reaching negative effects on living systems. This study aimed to investigate the toxic effects of Pb, through biochemical profiling and the ameliorative effects of quercetin against Pb-toxicity. Twenty-five male Wistar albino mice were divided into the following five groups. The CON-group received normal saline; the Pb-group received PbAc; the Pb + Q-CRN group received lead acetate followed by quercetin; the Q-CRN group received quercetin; and the CRN group received corn oil. After 4 weeks, the mice were euthanized. It was speculated that Pb significantly increased the levels of serine, threonine, and asparagine and decreased the levels of valine, lysine, and glutamic acid in the plasma of Pb-group, thus impairing amino acid metabolism. However, in the Pb + Q-CRN group, the level of these six amino acids was restored significantly due to the ameliorative effect of quercetin. The presence of lipid metabolites (L-carnitine, sphinganine, phytosphingosine, and lysophosphatidylcholine) in mice serum was confirmed by ESI/MS. The GPx, SOD, GSH, and CAT levels were significantly decreased, and the MDA level was significantly increased, thus confirming the oxidative stress and lipid peroxidation in the Pb group. The antioxidant effect of quercetin was elucidated in the Pb + Q-CRN group. Expression of CPT-I, CPT-II, LCAT, CROT, CACT, and MTR genes was significantly upregulated in the liver of Pb goup mice. Hence, the findings of this study proved that Pb exposure induced oxidative stress, upregulated gene expression, and impaired the lipid and amino acid metabolism in mice.

Keywords: amino acid metabolism; gene expression; lead acetate; lipid metabolism; quercetin.

FIGURE 1

Sources and molecular structure of quercetin.

FIGURE 2

Schematic representation of serum separation and sample pretreatment for metabolite acquisition by MS/MS.

FIGURE 3

Effect of intoxication of Pb on the liver (A) GSH, (B) SOD, (C) CAT, (D) GPx, and (E) MDA and the ameliorative effect of Que. The levels of GSH, SOD, CAT, GPx, and MDA were measured at the end of the experiment in the hepatic tissue homogenate. The level of significance was set at p < 0.05, and the data were analyzed using one-way ANOVA followed by Tukey’s test to compare all pairs of columns. The results were expressed as mean ± SD. Abbreviations: GSH: glutathione; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; MDA: malondialdehyde; CON: control group; Pb: lead group; Pb + Q-CRN: lead and quercetin blended in corn oil group; Q-CRN: quercetin blended in corn oil group; CRN: corn oil group; ANOVA: analysis of variance.

FIGURE 4

Bar diagram represents the effect of PbAc on mRNA expression of the (A) CPT I, (B) CPT II, (C) LCAT, (D) CROT, I CACT, and (F) MTR genes in different mice groups, i.e., CON, Pb, Pb + Q-CRN, Q-CRN, and CRN groups, respectively. The quantitative analysis was carried out at the end of the experiment of 28 days. The significance level was set at (p < 0.05) by using one-way ANOVA followed by Tukey’s test to compare all pairs of columns. Each error bar represents the mean ± SD. Abbreviations: CPT I carnitine palmitoyltransferase I; CPT II: carnitine palmitoyltransferase II; LCAT: lecithin–cholesterol acyltransferase; CROT: carnitine O-octanoyltransferase; CACT: mitochondrial carnitine/acylcarnitine carrier protein; MTR: 5-methyltetrahydrofolate-homocysteine methyltransferase; CON: control group; Pb: lead group; Pb + Q-CRN: lead and quercetin blended in corn oil group; Q-CRN: quercetin blended in corn oil group; CRN: corn oil group; ANOVA: analysis of variance.

FIGURE 5

Effect of intoxication of PbAc on area percent of (A) serine, (B) threonine, (C) asparagine, (D) valine, I(E) lysine, and (F) glutamic acid in the plasma of different mice groups, i.e., CON, Pb, Pb + Q-CRN, Q-CRN, and CRN groups, respectively. The quantitative analysis was carried out at the end of the experiment of 28 days. The significance level was set at (p < 0.05) by using one-way ANOVA followed by Tukey’s test to compare all pairs of columns. Each error bar represents the mean ± SD. Abbreviations: Ser: serine; Thr: threonine; Asn: asparagine; Val: valine; Lys: lysine; Glu: glutamic acid; CON: control group; Pb: lead group; Pb + Q-CRN: lead and quercetin blended in corn oil group; Q-CRN: quercetin blended in corn oil group; CRN: corn oil group ANOVA: analysis of variance.

FIGURE 6

MS/MS spectrum of L-carnitine, phytosphingosine, and sphinganine in negative and positive ion mode, respectively. Each m/z peak is circled by a color similar to the color of the border of its structure.

FIGURE 7

Full ms 2 and full ms 3 spectra of Lysophosphatidylcholine in positive ion mode. Each m/z peak is circled by a color similar to the color of the border of its structure.

FIGURE 8

Schematic representation of the functions of CPT I, CPT II, and CACT in mitochondria. CPT I is present at OMM. CPT I attaches a long-chain fatty acid, acyl CoA (palmitoyl-CoA and octadecenyl-CoA) to carnitine, and the resultant acylcarnitine is transported to the mitochondrial matrix by CACT. CACT is located at the IMM. Once inside the mitochondria, acylcarnitine is again converted to free carnitine and Acyl CoA. This free carnitine is again transported by CACT. The acyl CoA undergoes β-oxidation and is converted to acetyl CoA. The acetyl CoA can be converted into ketone bodies or enter the TCA cycle. CPT I: carnitine palmitoyltransferase I; CPT II: carnitine palmitoyltransferase II; CACT: mitochondrial carnitine/acylcarnitine carrier protein; OMM: outer mitochondrial membrane; IMM: inner mitochondrial membrane.

FIGURE 9

Illustrated the conversion of carnitine into acylcarnitine. This reaction is catalyzed by CPT I.

FIGURE 10

Illustrated the conversion of acylcarnitine into carnitine. This reaction is catalyzed by CPT II.

FIGURE 11

Illustrated the conversion of cholesterol into cholesteryl ester. This reaction is catalyzed by LCAT.

FIGURE 12

Pathway for the synthesis of methionine from S-adenosyl methionine. The MTR gene is responsible for synthesizing the enzyme methionine synthase that plays an important role in the synthesis of methionine amino acids.

FIGURE 13

Metabolic pathway of valine, serine, threonine, lysine, glutamic acid, and asparagine. After metabolism, the final product of these amino acids enters the tricarboxylic acid (TCA) cycle.