An Evaluation of Boar Spermatozoa as a Biosensor for the Detection of Sublethal and Lethal Toxicity

Abstract

A novel, objective, and rapid computed motility inhibition (CMI) assay was developed to identify and assess sublethal injury in toxin-exposed boar spermatozoa and compared with a subjective visual motility inhibition (VMI) assay. The CMI values were calculated from digital micrographic videos using a custom MATLAB® script by contrasting the motility index values of each experiment with those of the background and control experiments. Following a comparison of the CMI and VMI assays results, it was determined that their agreement depended on the shape of the dose-response curve. Toxins that exhibited a steep slope were indicative of good agreement between the assays. Those depicted by a gentle decline in the slope of the dose-response curve, the CMI assay were shown to be two times more sensitive than the VMI assay. The CMI assay was highly sensitive to the inhibition of mitochondrial function and glucose transport activity by sublethal doses of toxins and to disruption of cellular cation homeostasis by carrier ionophoric toxins, when compared to the cytotoxicity and lethal toxicity assays (i.e., that evaluated the inhibition of cell proliferation in somatic cell lines (FL, PK-15, and MNA cells)) and disruption to spermatozoa membrane integrity. The CMI assay recognized subtle sublethal toxicity changes in metabolism, manifested as a decrease in boar spermatozoa motility. Thus, it was feasible to effectively compare the objectively-measured numerical values for motility inhibition using the CMI assay against those reflecting lethal damage in the spermatozoa cells and somatic cell lines using a cytotoxicity assay.

Screening Mold Colonies by Using Two Toxicity Assays Revealed Indoor Strains of Aspergillus calidoustus Producing Ophiobolins G and K

“…The two screening assays detected sublethal toxins disturbing cellular energy metabolism (BSMI) as well as toxins affecting cellular proliferation (ICP) [18]. Screening of the biomass suspensions from individual colonies separated the ophiobolin-producing colonies by exhibiting a low toxic response (EC 50 < 2 µg mL −1 ) in both toxicity assays.…”

“…A total of 29 colonies displaying unique colony morphology in daylight were screened for toxicity using two complementary rapid screening tests. The tests measured (a) the toxins affecting cellular energy metabolism, mitochondria, and ion homeostasis as the inhibition of motility of boar spermatozoa (BSMI) [18,19,27] and (b) toxins affecting macromolecular synthesis and cytostatic activity as the inhibition of proliferation of a somatic cell line, PK-15 (ICP) [16]. The toxicity tests performed with the colonies from the primary isolation plates (Figure 1) were repeated with pure, single-spore cultures.…”

“…The assays measured sublethal toxicity in exposing resting boar sperm as BSMI after exposure for 30 min (rapid inhibition) and one day (slow inhibition), lethal toxicity as sperm membrane integrity damage (SMID), and cytostatic toxicity as ICP of PK-15 cells. The test protocols for the BSMI and SMID assays exposing the resting boar sperm cells are described in [17,18] and in [17] for the ICP assay. A randomly chosen colony, MH34, of the four most toxic colonies was identified at the species level, and the toxicity profiles were compared with the ethanol extracts of the selected indoor molds, commercial mycotoxins, and mycotoxins purified by the present authors.…”

“…The plate-grown biomass of the fungal pure cultures (100-300 mg) was extracted with ethanol, as described in [18]. The toxicity assays, the BSMI (boar sperm motility inhibition) assay after exposures of 30 min (rapid inhibition) and one day (slow inhibition), SMID (sperm membrane integrity damage) assay, and ICP (inhibition of cell proliferation) assay are all described in [17,18].…”

Penicillium expansum strain isolated from indoor building material was able to grow on gypsum board and emitted guttation droplets containing chaetoglobosins and communesins A, B and D

“…expansum are able to produce chaetoglobosins and communesins, which are secreted in membrane‐surrounded vesicles and liberated as liquid exudates (Salo et al ). Recently we showed that indoor Trichoderma strains produce toxic vesicles and guttation droplets containing peptaibols (Castagnoli et al ). The reported connection between exposure to toxins and weakened immune tolerance (Genius ; Tuuminen and Lohi ), as well as our preliminary findings aroused our interest in microbial toxigenesis and vesicle formation and guttation in indoor environments.…”