Metabolic Activation of Nonpolar Sediment Extracts Results in Enhanced Thyroid Hormone Disrupting Potency

Traditional sediment risk assessment predominantly considers the hazard derived from legacy contaminants that are present in nonpolar sediment extracts, such as polychlorinated biphenyls (PCBs), dioxins, furans (PCDD/Fs), and polyaromatic hydrocarbons (PAHs). Although in vivo experiments with these compounds have shown to be thyroid hormone disrupting (THD), in vitro their THD potency is not observed in nonpolar sediment extracts. This is hypothesized to be due to the absence of in vitro biotransformation which will result in bioactivation of the lipophilic compounds into THD hydroxyl metabolites. This study reveals that indeed metabolically activated nonpolar contaminants in sediments can competitively bind to thyroid hormone transport proteins. Sediment fractions were incubated with S9 rat microsomes, and the metabolites were extracted with a newly developed method that excludes most of the lipids to avoid interference in the applied nonradioactive 96-well plate TTR competitive binding assay. Metabolic activation increased the TTR binding potency of nonpolar fractions of POP-polluted sediments up to 100 times, resulting in potencies up to 240 nmol T₄ equivalents/g sediment equivalent (nmol T₄-Eq/g SEQ). This demonstrates that a more realistic in vitro sediment THD risk characterization should also include testing of both polar and medium polar sediment extracts for THD, as well as bioactivated nonpolar sediment fractions to prevent underestimation of its toxic potency

<i>Fusarium culmorum</i> wild types and ectopic transformants penetrate into wheat seeds and kill the seedling while Δ<i>FcStuA</i> mutants are unable to penetrate the seed.

<p>Seed viability after 3 days in the dark at 25°C on water agar (germination) after previous plug inoculation for 3 days with the two wild types (FcUK99 and Fc233B), the mutants (S12, S13 and S19), the ectopic transformants (S2 and S20) and a mock control inoculated with 10 µL water (W). Seeds were washed with NaClO before plating to eliminate external mycelium.</p

Growth of wild types, mutants and ectopic strains on different carbon sources obtained from various plant origins.

<p>Colony diameter in mm and standard deviation are indicated on the left and refer to the average of 3 biological replicates. The graph indicates the effect of carbon source on the growth ratio (mutant / wild type) after 5 days of growth on Petri dishes containing glucose, beechwood xylan, sugar beet arabinan, polygalacturonic acid, citrus pectin, apple pectin, potato azo-galactan or glutamic acid as a sole carbon source. Bars labelled with the same letter are not significantly different according to Tukey's post hoc test p<0.05. Bars represent SDs of the ratios (within each strain) of three biological replicates.</p

An <i>in vitro</i> comparison of the asexual development of the wild-type and Δ<i>FcStuA</i> mutant strains of <i>Fusarium culmorum</i>.

<p>Conidiogenesis by <i>StuA</i> deletion mutants, ectopic transformants and wild-type strains was observed with a light microscope (OLYMPUS BX41). Macroconidia of the wild-type FcUK99 strain (A) and the ectopic S20 strain (B) are formed from monophialides on branched conidiophores. Macroconidia of the Δ<i>FcStuA</i> mutant strains spores are generated directly from hyphae as in S12 and S13 (<i>StuA</i> mutants). (B) The same observations were made for <i>FcStuA</i> deletion mutant S19 obtained from strain Fc233B. Photos were taken with MOTICAM 2500 5.O MP live resolution (Motic). Hyphal diameter is smaller in the mutant.</p

Δ<i> </i>FcStuA mutants of <i>Fusarium culmorum</i> are unable to produce sporodochia.

<p>Sporodochia formation on SNA after 18 days growth. A: FcUK99, B: Fc233B; C: S12 (mutant obtained from A); D: S19 (mutant obtained from D).</p

The sensitivity of the mildly pathogenic wild-type Fc233B, ectopic and <i>FcStuA</i> deficient strains to various fungicides.

<p>Optical density of the <i>Fusarium culmorum</i> wild-type strain Fc233B, an ectopic (S20) and a <i>FcStuA</i> deficient strain (S19) in liquid cultures (200 µL, medium: 12.5% (w/w) potato dextrose broth) as affected by epoxiconazole (A), isopyrazam (B), tebuconazole (C), or trifloxystrobin (D) concentration after 5 days of incubation at 120 rpm and 22°C in the dark. An optical density close to 100% indicates no sensitivity to the fungicide, whilst an optical density close to 0% full inhibition. Error bars represent the standard error of 3 replicates.</p

Pathogenicity of the <i>Fusarium culmorum</i> wild type and Δ<i>FcStuA</i> mutant strains and ectopic transformants on various plant species and tissue types.

<p>A: 21-d old durum wheat cv Claudio seedlings infected with Δ<i>FcStuA</i> mutants, wild types and ectopics and mock inoculated (test). No FRR symptoms were detected on the plants derived from the seeds inoculated with the mutants (S19, S20, S12 and S13). B: Spike of durum wheat cv Simeto infected with FcUK99, with an ectopic transformant or with the four independent Δ<i>FcStuA</i> mutants. No symptoms are visible on the mutant inoculated spikes. Photographs were taken 14 days post inoculation. C: Testing the ability for the same set of fungal strains to colonise three different plant tissues: apple (1), potato (2) and tomato (3). Cut or wounded surfaces were inoculated with <i>StuA</i> deleted mutants (S12, S13, S19), ectopic transformants (S2 and S20) or the wild-type strains (FcUK99 and Fc233B). Photographs were taken at either 4 days post inoculation (dpi) (apple and tomato) or 7 dpi (potato).</p

The sensitivity of the highly pathogenic wild-type FcUK99, ectopic and <i>FcStuA</i> deletion mutants to various fungicides.

<p>The optical density of liquid cultures of the <i>Fusarium culmorum</i> wild type strain FcUK99, an ectopic and <i>FcStuA</i> deficient strains (S9, S12, S13) growing in 200 µL of 12.5% (w/w) potato dextrose broth when augmented with different concentrations of fungicides (A) epoxiconazole, (B) isopyrazam, (C) tebuconazole, or (D) trifloxystrobin. The optical density measurements were made after 5 days of incubation at 120 rpm and 22°C in the dark. An optical density close to 100% indicates no sensitivity to the fungicide, whilst an optical density close to 0% full inhibition. Error bars represent the standard error of 3 replicates.</p

Primer sequences used to obtain the transforming constructs and to identify mutants by PCR and Southern blotting.

<p>Primer sequences used to obtain the transforming constructs and to identify mutants by PCR and Southern blotting.</p

Scanning electron microscope analysis of the infection of durum wheat seeds by <i>Fusarium culmorum</i> wild-type and Δ<i>FcStuA</i> mutant strains.

<p>These ZEISS EVO LS images reveal the appearance of the surface of a seed of durum wheat placed over a mycelium disk of either wild-type <i>Fusarium culmorum</i> FcUK99 (A and B) or its ΔFc<i>StuA</i> mutant (C and D), 3 days after incubation at 25°C. During observation the samples were subjected to the following conditions: temperature 2°C, humidity 85% and pressure 600 Pa. A and C panel show an apical seed view. B and D show a detail of the mycelium surrounding the seed.</p