Allyl isothiocyanate that induces GST and UGT expression confers oxidative stress resistance on C. elegans, as demonstrated by nematode biosensor.

BACKGROUND: Electrophilic xenobiotics and endogenous products from oxidative stresses induce the glutathione S-transferases (GSTs), which form a large family within the phase II enzymes over both animal and plant kingdoms. The GSTs thus induced in turn detoxify these external as well as internal stresses. Because these stresses are often linked to ageing and damage to health, the induction of phase II enzymes without causing adverse effects would be beneficial in slowing down ageing and keeping healthy conditions. METHODOLOGY/PRINCIPAL FINDINGS: We have tested this hypothesis by choosing allyl isothiocyanate (AITC), a functional ingredient in wasabi, as a candidate food ingredient that induces GSTs without causing adverse effects on animals' lives. To monitor the GST induction, we constructed a gst::gfp fusion gene and used it to transform Caenorhabditis elegans for use as a nematode biosensor. With the nematode biosensor, we found that AITC induced GST expression and conferred tolerance on the nematode against various oxidative stresses. We also present evidence that the transcription factor SKN-1 is involved in regulating the GST expression induced by AITC. CONCLUSIONS/SIGNIFICANCE: We show the applicability of the nematode biosensor for discovering and evaluating functional food substances and chemicals that would provide anti-ageing or healthful benefits.

Acrylamide-responsive genes in the nematode Caenorhabditis elegans.

As acrylamide is a known neurotoxin for many animals and potential carcinogen for humans, it came as a surprise when the Swedish National Food Agency and Stockholm University reported in 2002 that it is formed during the frying or baking of foods. We report here genomic and proteomic analyses on genes and proteins of Caenorhabditis elegans exposed to 500 mg/l acrylamide. Of the 21,120 genes profiled, 409 genes were more than twofold upregulated and 111 genes were downregulated. Upregulated genes included many that encode detoxification enzymes such as glutathione S-transferases (GSTs), uridine diphosphate-glucuronosyl/glucosyl transferases, and short-chain type dehydrogenases but only one cytochrome P450. Subsequent proteomic analysis confirmed the heavy involvement of GSTs. Because of their high expression levels and central roles in acrylamide metabolism, we analyzed the in vivo expression patterns of eight gst genes. Although all encoded GST and were more than twofold upregulated by acrylamide treatment, their expression patterns were varied, and their regulation involved the transcription factor SKN-1 (a C. elegans homolog of Nuclear factor E2-related factors 1 and 2). We then selected the gst-4::gfp-transformed C. elegans to study the detoxification rate of acrylamide and its metabolite glycidimide in living animals. This animal detects acrylamide as a green fluorescence protein (GFP) expression signal in a dose- and time-dependent manner and may prove to be a useful tool not only for rapidly and inexpensively detecting acrylamide, a harmful substance in food, but also for analyzing mechanisms of GST induction by acrylamide and other inducers like oxidative stresses.

A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food.

By DNA microarray and protein 2-DE screens for Caenorhabditis elegans genes up-regulated by acrylamide, we selected the gst-4 gene and constructed a gst::gfp fusion gene, which was used to transform C. elegans into a biosensor for acrylamide. This biosensor detects acrylamide as a GFP-expression signal in a dose- and time-dependent manner. When the biosensor was exposed to acrylamide together with commercially available powdered green tea, GFP levels decreased to the control level, suggestive of acrylamide detoxification or prevention of GST induction. The present methodology should be applicable for screening of not only harmful substances but also substances that reduce or counteract their harmfulness or action, with appropriately constructed visible biosensors.

Extremely low dose of acrylamide decreases lifespan in Caenorhabditis elegans.

The neurotoxic, genotoxic and carcinogenic effects of industrial exposure to acrylamide have been studied in animals and humans for more than 30 years. A recent search for the cause of high background levels of acrylamide in industrially unexposed people revealed that it is formed during the frying or baking of foods by means of the Maillard reaction. To evaluate the biological consequences of continuous exposure to acrylamide at levels found in common foodstuffs, we studied the effects of acrylamide on the three parameters of (1) growth, (2) fecundity and (3) lifespan in the nematode Caenorhabditis elegans. As for growth and fecundity, no deleterious consequences were observed when the animals were raised in the acrylamide concentrations of 0.5 microg/L to 5mg/L, which are commonly found in daily consumed foodstuffs. Conversely, in 500 mg/L of acrylamide, they showed retarded growth with reduced body and brood sizes. Unlike the first two parameters, however, lifespan decreased significantly even in 0.5 microg/L of acrylamide, the maximum theoretical concentration allowed in drinking water by the WHO and US EPA. Very interestingly, the acrylamide effect on lifespan is biphasic as lifespan increased to near normalcy in 5 mg/L and decreased again in 500 mg/L.

Early embryogenesis of the pinewood nematode Bursaphelenchus xylophilus.

The early embryogenesis and cell lineage of the pinewood nematode Bursaphelenchus xylophilus was followed from a single-cell zygote to a 46-cell embryo under Nomarski optics, and elongation of the microtubules was studied by immunostaining. As a B. xylophilus oocyte matures, it passes through a passage connecting the oviduct with the quadricolumella, the distal part of the uterus, and reaches the quadricolumella where it stays for a few minutes and is fertilized. After fertilization, the germinal vesicle disappears, an eggshell is formed, and the male and female pronuclei appear. The pronuclei move toward each other and fuse at the center of the egg. Around this time, the microtubule-organizing center appears. The presumptive region of sperm entry into the oocyte becomes the future anterior portion of the embryo. This anterior-posterior axis determination is opposite to that of Caenorhabditis elegans, where the sperm entry site becomes the posterior portion of the embryo. The optimal growth temperatures of these two nematodes also differ in that temperatures of about 30 degrees C afford the fastest growth rate and highest hatching frequency in B. xylophilus. Otherwise, the lineage resembles that of C. elegans with respect to timing, positioning and the axis orientation of each cell division.