Uptake and subcellular distribution of aflatoxin B 1 by excised, cultured soybean roots and toxin effects on root elongation

Abstract

Aflatoxins (AFTs), potent hepatocarcinogens, mutagens and teratogens, are secretions of Aspergillus flavus and A. parasiticus which can grow upon Glycine max beans. As previously reported, AFTs can inhibit both the elongation of excised soybean roots and uptake as well as incorporation of [ 14C]-leucine by and into cultured roots. This suggests that the AFTs may gain entrance into the roots. Here, we report both the uptake and distribution of aflatoxin B 1 (AFB 1) by and within the roots as well as the effects of the toxin upon time-dependent changes in root dry weight. Excised roots (from 3-day-old imbibed seeds) were incubated 18 hr with either 30 μg/ml unlabeled AFB 1 or 695,381 dpm [ 3H]-AFB 1 and subcellular fractions (1000, 40,000 and 80,000 g ) pellets and 80, 000 g supernatant) prepared by differential centrifugation of Miracloth filtrates of root homogenates. Both growth media and resuspended fractions were partitioned against chloroform and the organic phase chromatographed upon Adsorbosil + 1 soft and hard thin layer chromatoplates in chloroform: acetone: water (8.0 + 12.0 + 1.5). The separated unlabeled and labeled AFB 1 were quantified by u.v.-spectroscopy at 362 nm and liquid scintillation counting, respectively, after their removal from the chromatoplates. Inclusion of AFB 1 within the growth medium inhibited the time-dependent increase in root dry weight which occurred during culturing. Upon completion of culturing, roots took-up 24–66% of the AFB 1 within the growth medium. Of the AFB 1 taken up, 5.9–8.6, 9.5–10.3, 2.4–9.1 and 60.7–77.1% were found in the 1000, 40,000 and 80,000 g pellets and as well as the 80,000 g supernatant, respectively, which were obtained by centrifugation. Based upon limited marker enzyme analyses and electron microscopy as well as previous chemical data, the organelle contents of the fractions were tentatively identified as 1000 (nuclei with nuclear envelope-endoplasmic reticulum connections), 40,000 (dictyosomes-mitochondria with nuclie and plasmalemma) and 80,000 (plasmalemma with mitochondria, dictyosomes and endoplasmic reticulum) g pellets and 80,000 g supernatant (ribosomes and cytosol). Thus, most of the AFB 1 which taken-up by roots, remains in a presumed ribosome-cytosol fraction.