ISB News Report
Caius M Rommens
SEPTEMBER 2008
Some of our most popular processed foods contain small amounts of toxic acrylamide. Currently available methods that lower the accumulation of this reactive compound have a negative effect on sensory characteristics and/or are not broadly applicable. Realizing their limited options, several food companies have committed to substantially reducing the acrylamide levels in fried and baked potato products over the next three years. A novel method that could be applied to produce the desired low-acrylamide foods was recently published in Plant Biotechnology Journal . According to this method, potato plants are transformed with an all-native silencing construct that targets two asparagine synthetase genes. The resulting plants produce tubers with very low levels of the acrylamide precursor asparagine. French fries and potato chips from these “intragenic” plants contain up to 20-fold lower levels of acrylamide than their untransformed counterparts. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties by the low-asparagine potatoes would reduce the average daily intake of acrylamide by almost one-third.
Acrylamide is a typical constituent of modern diets and may play a minor role in the emergence of certain
“modern” diseases. Dietary intake levels of acrylamide have been rising in the Western world since the early 1900s and are currently estimated at 40 µg/person/day. Acrylamide is formed in starchy foods when they are baked or fried as a consequence of the reaction between certain sugars and the free amino acid asparagine ( Fig. 1 ). This reactive compound is present in products derived from wheat flour, coffee beans, and potato tubers. Ingested acrylamide is readily absorbed and, in part, metabolized by a cytochrome P450 to produce mercapturic acid and glycidamide. Whereas mercapturic acid is excreted via urine, both the remaining acrylamide and its reactive metabolite bind to various proteins as well as
DNA. High levels of adduct formation have been linked to animal health issues, including cumulative nerve terminal damage. As suggested by several recent studies, acrylamide intake may also be associated with the increased incidence of neurodegenerative and other types of diseases 1-3 .
Figure 1 Toxicity of heat-induced acrylamide formation in starchy foods ( A ) Nitrogen is acquired from the environment via nitrate reduction or ammonia uptake, and assimilated through the action of enzymes such as glutamine synthetase (GS), ferredoxin-dependent glutamate synthase (GOGAT), aspartate aminotransferase (ASPAT), asparagine synthetase (AS), asparaginase (ASPASE), and glutamate dehydrogenase
(GDH). GLN = glutamine, αKG = α-ketoglutarate, OXA = oxaloacetate, ASP = aspartate. ( B ) Asparagine plays a role in the long-distance transport of nitrogen, and in the storage of this compound in sink organs such as tubers and seeds. ( C ) At temperatures exceeding 120 O C, the α-NH
2
group of asparagine reacts with the carbonyl group of reducing sugars to produce a Schiff base which, especially under high moisture conditions, forms the
Amadori compound N-(D-glucos-1-yl)-L-asparagine. This unstable compound then forms acrylamide through decarboxylative deamination. ( D ) Upon intake of processed starchy foods, acrylamide is readily absorbed and distributed among tissues. Some acrylamide is detoxified whereas another part is converted to glycidamide. Both acrylamide and glycidamide bind DNA and proteins to form adducts. This adduct formation is linked to various diseases.

ISB News Report SEPTEMBER 2008
Attempts to lower the acrylamide levels in foods are either not broadly applicable or negatively affect food color, texture, and taste.
Recently developed methods that limit acrylamide formation to some extent require changes in grower or processor practices. Such modifications often limit the applicability of the method and/or affect the quality of the final product. For instance, the beneficial effect of sulfur fertilization on lowering the acrylamide potential of potato and wheat is off-set by increased farmer input costs and sulfur contamination issues. Furthermore, the partial reduction in acrylamide concentration that can be achieved by modifying processing variables, such as the time and temperature of heating, yields products with altered sensory characteristics. A third approach incubates raw materials with either asparagine-metabolizing enzymes or amino acids that compete with asparagine in the Maillard reaction. Such treatments are only partially effective for some raw food ingredients, require high concentrations of the additive, and are too difficult and costly to apply broadly.
Inhibition of the starch degradation pathway in potato tubers lowers their acrylamide potential by up to four-fold.
A more preferred route to lowering the accumulation of acrylamide is to shift to crops that are naturally poor in acrylamide precursors. Although there are currently no such varieties available that also display all the additional input, processing, and quality traits demanded by the processing industry, it is possible to reduce the acrylamide potential of existing varieties through “intragenic” modification 4 . This new extension of plant breeding modifies the plant’s own genome through transformation with native genetic elements. An initial approach was based on the targeted inhibition of the starch degradation process 5 . Cold-stored potato tubers express several genes encoding starch degradation enzymes such as water dikinase R1 and amyloplast-targeted phosphorylase-L. Tuber-specific silencing of these two genes lowers cold-induced sweetening without negatively affecting plant phenotypes. French fries from the intragenic tubers accumulate only about 25 – 35% of the acrylamide that is produced in untransformed tubers. Fortuitously, they also contain a higher level of starch while displaying increased visual and sensory characteristics.
Low-acrylamide French fries and potato chips from low-asparagine potato tubers more effective method silences two asparagine synthetase genes in tubers 6
. A second and even
. Whereas controls contain large amounts of asparagine (160 mg/100 g FW), tubers of transformed “pSIM1256” lines accumulate only ~10 mg/100 g FW. The dramatic decrease in asparagine levels is associated with slightly elevated levels of glutamine but does not affect the production of other amino acids, and also does not alter total protein yield. Both French fries and potato chips from the intragenic tubers accumulate much less acrylamide than is present in controls ( Fig. 2 and data not shown). This modification does not alter the color, texture, and taste of the final product. Furthermore, preliminary greenhouse data indicate that intragenic lines display the same agronomic features as their untransformed counterparts. If these results can be confirmed by follow-up studies from field-grown tubers, all-native fry and chip products with very low levels of acrylamide could be offered as a new market choice within the next five years. Given the important role of processed potato products in the Western diet, a replacement of current varieties with intragenic potatoes would reduce the average daily intake of acrylamide by almost one-third. Even greater reductions could be accomplished by applying the described methods to wheat and coffee as well.

ISB News Report SEPTEMBER 2008
Figure 2 Acrylamide levels in processed potato products Data are expressed as percentage of the control for French fries ( A ) and potato chips ( B ), and represent the mean of three measurements ± SD. Asterisks indicate significant differences with the controls (P<0.05).
References
1. Hogervorst JG, Schouten LJ, Konings EJ, Goldbohm RA, van den Brandt PA. 2007. A prospective study of dietary acrylamide intake and the risk of endometrial, ovarian, and breast cancer. Cancer Epidemiol. Biomarkers Prev.
16 , 2304-2313
2. LoPachin RM, Barber DS, Gavin T. 2008. Molecular mechanisms of the conjugated alpha,beta-unsaturated carbonyl derivatives: relevance to neurotoxicity and neurodegenerative diseases. Toxicol. Sci.
104 , 235-249
3 Olesen PT, Olsen A, Frandsen H, Frederiksen K, Overvad K, Tjønneland A. 2008. Acrylamide exposure and incidence of breast cancer among postmenopausal women in the Danish Diet, Cancer and Health Study. Int. J. Cancer 122 , 2094-2100
4. Rommens CM, Haring MA, Swords K, Davies HV, Belknap WR. 2007. The intragenic approach as a new extension to traditional plant breeding. Trends Plant Sci.
12 , 397-403
5. Rommens CM, Ye J, Richael C, Swords K. 2006. Improving potato storage and processing characteristics through all-native DNA transformation. J. Agric. Food Chem.
54 , 9882-9887
6. Rommens CM, Yan H, Swords K, Richael C, Ye J. 2008. Low-acrylamide French fries and potato chips. Plant Biotechnol J.
, DOI:
10.1111/j.1467-7652.2008.00363
Caius M Rommens
J. R. Simplot Company, Simplot Plant Sciences
Boise, ID 83706, USA crommens@simplot.com