In vitro study of AFB₁ and AFM₁ effects on human lymphoblastoid Jurkat T-cell model

Figures & data

Table 1. Primer sequences.

Primer sequences	Length
L-32 Sense 5′-CCT CAG ACC CCT TGT GAA GC-3′	145 bp
Anti-sense 5′-GCC CTT GAA TCT TCT ACG AAC C-3′
IL-2 Sense 5′-AAC TCA CCA GGA TGC TCA CAT TTA-3′	148 bp
Anti-sense 5′-TTC CTG GGT CTT AAG TGA AAG TTT-3′
IFNγ Sense 5′-TCA GCT CTG CAT CGT TTT GG-3′	120 bp
Anti-sense 5′-GTT CCA TTA TCC GCT ACA TCT GAA-3′
IL-8 Sense 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′	289 bp
Anti-sense 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′

Figure 1. Effects of AFB₁ and AFM₁ on Jurkat cell proliferation. Jurkat T-cells were incubated in the presence of increasing concentrations (3–50 μM) of AFB₁ (black bar) or AFM₁ (grey bar) for 24 h. Eighteen hours prior to harvesting, cells were pulsed with 1 µCi/well [³H]-thymidine. DNA synthesis was then measured by [³H]-thymidine incorporation and expressed as counts per minute (cpm). Control (ctr): cells incubated with medium containing ethanol (0.1%) only. Each value represents the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 versus ctr.

Figure 2. Effects of AFB₁ and AFM₁ on Jurkat cell caspase-3 activity and LDH release. (a) Each end-point was analyzed following exposure to AFB₁ (black bar) or AFM₁ (grey bar) for 24 h. Control (ctr) cells were incubated with medium containing ethanol (0.1%) only. In (a), cells treated with 10 μM actinomycin D (ActD) served as positive control. Values were ultimately expressed as nmole AMC/mg protein in cell lysate. In (b), cells treated with 1% NP-40 served as positive control. Amount of LDH released into medium was expressed as a percentage of LDH released from positive control. All values represent mean ± SD of three independent experiments. ***p < 0.001 versus ctr.

Figure 3. Effects of AFB₁ and AFM₁ on the Jurkat T cell cytokine expression. mRNA levels of select cytokines in Jurkat cells following 24-h incubation with PMA/ionomycin concurrent with increasing concentrations (3–50 μM) of AFB₁ (black bar) or AFM₁ (grey bar). Control (ctr) was unstimulated cells; the positive control (ctr+) was cells incubated with PMA/ionomycin. Unstimulated cells were incubated with medium containing ethanol (0.1%) only. mRNA levels of (a) IFNγ, (b) IL-2, (c) IL-8, and L-32 were subsequently assessed by real-time PCR analysis. Results represent mean ± SD of two independent experiments. ***p < 0.001 versus ctr+.