Effects of aflatoxin and fumonisin on gene expression of growth factors and inflammation-related genes in a human hepatocyte cell line

Abstract

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are mycotoxins widely distributed in maize and maized-based products, often occurring together. The implications of co-exposure to aflatoxin and fumonsin for human health are numerous, but a particular concern is the potential of FB1 to modulate AFB1 hepatotoxicity. This study evaluated the toxicity of these mycotoxins, alone or combined, in a human non-tumorigenic liver cell line, HHL-16 cells, and assessed the effects of AFB1 and FB1 on expression of genes involved in immune and growth factor pathways. The results demonstrated that in HHL-16 cells, both AFB1 and FB1 had dose-dependent and time-dependent toxicity, and the combination of them showed a synergistic toxicity in the cells. Moreover, AFB1 caused upregulation of IL6, CCL20, and BMP2, and downregulation of NDP. In combination of AFB1 with FB1, gene expression levels of IL6 and BMP2 were significantly higher compared to individual FB1 treatment, and had a tendency to be higher than individual AFB1 treatment. This study shows that FB1 may increase the hepatoxicity of AFB1 through increasing the inflammatory response and disrupting cell growth pathways.

Keywords: HHL-16 cells; aflatoxin; fumonisin; gene expression; toxicity.

Figure 1.

Single AFB₁ (A) or FB₁ (B) treatment on HHL-16 cells for 24 and 48 h. Control: untreated cells; D: DMSO treatments; A: AFB₁ treatments; P: PBS treatment; F: FB₁ treatment. DMSO and PBS treatments were normalized against the control group. AFB₁ treatments were normalized against corresponding DMSO treatments (i.e. A1 against D1), and FB₁ treatments were normalized against the PBS treatment. With the increasing of either AFB₁/FB₁ concentrations or treated time, the cell viability was decreased, so there were dose-dependent and time-dependent cytotoxicity of both AFB₁ and FB₁ in HHL-16 cells. Data are presented as mean ± SEM, and the results were repeated three times.

Figure 2.

(A) Combined treatment of AFB₁ and FB₁ on HHL-16 cells for 24 and 48 h. Control: untreated cells; D: DMSO treatment; P: PBS treatment; A: AFB₁ treatment; F: FB₁ treatment; PBS and DMSO treatment were normalized against control groups. Combined treatments were normalized against corresponding combined treatment of DMSO and PBS. Expected additive effect: the sum of the toxic effect of single AFB₁ and FB₁. Combined AFB₁ and FB₁ treatment has a synergistic effect on HHL-16 cells 24 and 48 h post the treatment, it is both dose-dependent and time-dependent manner. Data was presented as mean ± SE, and the results were repeated three times. (B) Combinatory effects of aflatoxin and fumonisin evaluated by the combination index (CI) theorem. Fa: fraction affected (Fa = 1 − inhibition of cell viability%)/100. CI < 1, =1, >1 represent for synergistic, additive, and antagonistic effects, respectively. A synergistic effect was observed in the combination of AFB₁ and FB₁ in HHL-16 cells.

Figure 3.

Gene expression of cytochrome P450 (CYP) enzymes in HHL-16 cells exposed to aflatoxin for 24 h. Control: untreated cells; D: DMSO treatment; A: aflatoxin treatment. ACTB: Beta-actine, a reference gene used for the normalization to CYP1A2 (A), CYP3A4 (B) and CYP3A5 (C). A significant increase of CYP3A4 and CYP3A5 were observed in HHL-16 cells after aflatoxin treatment for 24 h. Although there is a tendency to be higher expression of CYP1A2, it did not reach significant level.

Figure 4.

Heat map of gene expression fold change of several selected genes in growth factors pathway in HHL-16 cells exposed to single AFB₁ (10 µg/ml) or single FB₁ (50 µg/ml) for 24 h. A: AFB₁ treatment; F: FB₁ treatment. The colours represent the up-regulated (black) and down-regulated (white) genes compared to the control group, respectively.

Figure 5.

Differential gene expression of candidate genes, IL6 (A), CCL20 (B), BMP2 (C), and NDP (D), involved in immune and growth factors pathways in HHL-16 cells exposed to AFB₁ for 24 h. Control: untreated cells. D: DMSO treatments. A: AFB₁ treatments. GAPDH and ACTB, two reference genes were used to normalize the gene expression of IL6, CCL20 and NDP. Dose-dependent increasing of IL6, CCL20 and BMP2, and dose-dependent decreasing of NDP were observed.

Figure 6.

Differential gene expression of candidate genes, IL6 (A), CCL20 (B), BMP2 (C), and NDP (D), involved in immune and growth factors pathways in HHL-16 cells exposed to both AFB₁ and FB₁ for 24 h. Control: untreated cells. D: DMSO treatments. A: AFB₁ treatments. F: FB₁ treatments. GAPDH and ACTB, two reference genes were used to normalize the gene expression of IL6, CCL20, BMP2 and NDP. Dots, diagonal lines, crosshatch, and black columns represent the control, single AFB₁, single FB₁, and combined AFB₁ and FB₁ treatment, respectively. IL6 and BMP2 expression levels were significantly higher after the combined treatment of 5 µg/ml AFB₁ and 100 µg/ml FB₁ than 100 µg/ml FB₁ treatment alone and had a tendency to be higher compared to the individual 5 µg/ml AFB₁ treatment.