INFORMATION SYSTEMS FOR BIOTECHNOLOGY


September 2006
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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IN THIS ISSUE:



A NEW CONCEPT TO ENGINEER SPECIFIC TRANSCRIPTION OF TRANSPLASTOMIC GENES
Silva Lerbs-Mache

Plants are increasingly becoming attractive bioreactors for production of commercially important proteins. Plastid transformation, which has been recently developed for this purpose, offers several advantages. (1) Each molecular unit of the chloroplast genome has a relatively small size, ca. 150 kbp, which facilitates homologous recombination and hence permits introduction of DNA modifications such as deletions or insertions at precise locations into the chloroplast chromosome. This is in contrast with the random insertion of DNA fragments into the nuclear genome obtained using the well-known method of gene transfer using Agrobacterium tumefaciens. (2) The overall organization of the chloroplast genome is of prokaryotic type, and therefore complex phenomenon such as position effects and epigenetic controls encountered in nuclear gene regulation do not exist. (3) The chloroplast chromosome has a large number – up to one hundred – of copies per mature chloroplast. For example, 10 000 chromosome copies per cell are present in mesophyll, which have approximately 100 chloroplasts/cell. Consequently, the amount of heterologous protein corresponding to a given transgene can reach a relative high level, e.g., in some cases up to 45% of total soluble proteins.1 (4) Finally, most plant species of agronomic interest do not transmit the chloroplast genome by pollen, as their chloroplasts are maternally inherited. Maternal inheritance avoids gene dispersion and transfer to other, non-transformed, related plant species.

Application of plastid transformation technology is limited by the difficulty of obtaining regulated, selective expression of the transgenes. Except for the blue light-inducible psbD promoter, there are no specific inducible endogenous promoters available; hence transgenes are constitutively expressed throughout plant development. As a consequence, plant growth is often heavily disturbed by the noxious effect of overproduction of the transgene product.

To attempt to solve this problem, a nuclear DNA-encoded and plastid-targeted T7 phage RNA polymerase gene was introduced into tobacco and placed under control of a T7 promoter. This system is specific and inducible since there are, a priori, no T7 promoter sequences on the plastid genome, and inducible nuclear promoters are available for expression of the T7 RNA polymerase. However, it turns out that, although transgene mRNA accumulated to a high level, the corresponding protein was not detectable, and plants grew slowly or died soon after germination.2 The reason for this incompatibility of the phage T7 transcription system with the plastid gene expression machinery is not yet clear. In addition to the prokaryotic type plastid encoded RNA polymerase (PEP) transcription system, transcription in plastids also involves nucleus encoded RNA polymerases (NEP) that are similar to the T7 RNA polymerase.3 Although NEP promoters are different from T7 promoters, cross-reaction of the two transcription systems has been reported and might account for the disturbance in growth of plants transformed by T7 RNA polymerase.

Another approach to controlled transgene expression in chloroplasts uses the bacterial lac repressor protein, which prevents transcription of the transgene until repression is relieved by spraying plants with isopropyl-b-D-galactopyranoside (IPTG).4 Because of the use of spray, this technique is limited to the greenhouse-grown plants and to transgene expression in aerial tissues.

We have now developed a method based on the same principle as the T7 system, i.e., a double transformation, but it does not introduce a new RNA polymerase into plastids. Our approach is based on the modification of an endogenous transcription factor to make it specific for a foreign promoter that does not exist on the plastid genome. The specificity of promoter recognition in bacteria is accomplished by transcription initiation factors, so-called sigma factors. In Escherichia coli, the s70 factor first interacts with the core RNA polymerase (consisting of four core subunits: 2a, b and b') forming a holoenzyme able to recognize the specific promoter elements.

Sigma factors are composed of C-terminal parts that recognize the promoter elements of the template DNA and N-terminal parts that recognize the core RNA polymerase. This simplified scheme is also applicable to PEP, which has preserved the prokaryotic traits of its cyanobacterial origin. PEP is composed of five subunits instead of four, because the b' subunit is split into two parts, b' and b", as in cyanobacteria.

Specificity of promoter recognition is also enabled by sigma-like factors. Six different sigma-like factor genes are present in the nuclear genome of Arabidopsis thaliana, and all possess characteristics of the s70 family of E. coli. They are also composed of a C-terminal DNA recognition domain and an N-terminal RNA polymerase interaction domain. The C-terminal parts of the plant sigma-like proteins that recognize the promoter regions are well preserved. Analogous to E. coli sigma factors, they can be subdivided into regions 2.4 and 4.2, necessary for promoter recognition, region 2.3, implicated in DNA melting, and regions 2.1 and 3.2, involved in RNA polymerase-core interaction. However, the N-terminal parts of the plant sigma-like proteins are different from the corresponding part of the E. coli s70 factor, resulting in functional diversity, i.e., specific interaction with only the PEP or the E. coli core enzyme. This specificity could be demonstrated by functional complementation of E. coli s70 thermosensitive mutants. Only a fusion protein, consisting of the C-terminal part of the plant SIG2 protein fused to the N-terminal part of E. coli s70 factor, was able to fully complement the mutant.5

Based on this result, we have created a hybrid (plant - E. coli) transcription system in which the plastid transgene is placed under control of the E. coli heat shock promoter groE, and specific transcription of the transgene is achieved by supplying a chimeric transcription factor composed of the N-terminal part of plant sigma factor 1(SIG1) and the C-terminal part of the E. coli heat shock transcription factor s32 (6, see Figure 1). Because of its C-terminal part, the hybrid transcription factor SIG1/s32 should recognize only the gene driven by the groE promoter on the plastid genome; and because of its N-terminal part, the factor should be able to interact with PEP.

E. coli heat shock promoters are very different in sequence from s70 promoters, and s32 confers to RNA polymerase the ability to initiate transcription exclusively at heat-shock genes. The s32 holoenzyme is unable to recognize promoters that are transcribed by the principal s70 holoenzyme, and the s70 holoenzyme does not recognize heat shock gene promoters. Importantly, s32 promoters are absent on the chloroplast genome, i.e., the introduction of a transgene driven by a s32 promoter should be expressed with high specificity.

Thus, a transgene coding for E-YFP (Enhanced Yellow Fluorescent Protein) directed by the E. coli groE heat shock promoter was introduced into the tobacco chloroplast genome using polyethylene glycol-mediated plastid transformation. Transplastomic tobacco plants were obtained for which all chloroplast chromosomes were transformed (homoplastomic plants). The hybrid sigma factor SIG1/s32 was finally expressed by transient transformation via Agrobacterium tumefaciens of young transplastomic plants using the construct described above.

Results show specific expression of the E-YFP protein in the homoplastomic plants after transient transformation, thus confirming our assumption that, by using this hybrid transcription system, a transgene could be specifically expressed in chloroplasts. In future work the construct coding for the hybrid sigma factor should be introduced into the transplastomic plant by stable transformation under the control of a developmentally regulated promoter. Thus, a transplastomic gene of interest could be expressed at high level at a specific stage of development.

References

1. De Cosa et al. (2001) Over expression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nature Biotechnology 19, 71-74

2. Magee AM et al. (2004) T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype. Transgenic Research 13, 325-337

3. Toyoshima Y et al. (2005) Plastid Transcription in Higher Plants. Crit. Rev. Plant Sci. 24, 59-81

4. Mühlbauer SK and Koop H-U (2005) External control of transgene expression in tobacco plastids using the bacterial lac repressor. Plant J. 43, 941-946

5. Hakimi MA et al. (2000) Evolutionary conservation of C-terminal domains of primary sigma-70 type transcription factors between plants and bacteria. Journal Biological Chemistry 275, 9215-9221

6. Buhot L et al. (2006) Hybrid transcription system for controlled plastid transgene expression. Plant Journal 46, 700-7007

 

Silva Lerbs-Mache
Laboratoire Plastes et Différenciation cellulaire
Universitè Joseph Fourier
Grenoble Cedex 9, France
Silva.Lerbs-Mache@ujf-grenoble.fr



LEGISLATING AGBIOTECH
Phillip B C Jones

In 2005, the majority of bills adopted by state legislators supported agbiotech. The Pew Initiative on Food and Biotechnology announced this discovery in June. The Initiative also released their updated fact sheet and web database on state and federal legislative activity in the agbiotech area.

Pew Initiative analysts counted 117 pieces of legislation related to agricultural biotechnology introduced in 33 states and the District of Columbia during the 2005 legislative session. Hawaiian legislators had been the busiest; they introduced 33 bills, or 28 percent of the total. Legislators in the Northern Plains and Midwest introduced 23 percent of the bills; Northeast legislators generated 22 percent; the South 16 percent; the West 9 percent; and Alaska 2 percent.

One of the most active legislative topics in 2005 also represented a new trend: bills to preempt or disallow local and county initiatives aimed at limiting or prohibiting genetically engineered (GE) seeds and crops. An example of a local effort took place in June when Santa Cruz County supervisors approved a moratorium on growing GE crops within county lines. The prohibition should have little impact on Santa Cruz County agriculture; no GE crops are grown here. Berries and lettuce dominate the county’s crops, rather than the typical targets of genetic engineering: corn, cotton and soybeans.

Santa Cruz County joined Trinity, Marin, and Mendocino counties, which also prohibit the planting and production of GE crops. Local bans have provoked state legislators who do not want to see California become a patchwork of disparate regulations. Senate Bill 1056 would overrule attempts by local jurisdictions to regulate crops. "The goal of this legislation is simply to say that we have a consistent policy," explained Sen. Dean Florez (D-Shafter), SB 1056’s author. "Twelve other states have passed laws like this." Pew Initiative analysts anticipate that preemption will continue to be an active issue for state legislatures throughout this year.

Legislation introduced in 2005 also focused on the peaceful coexistence of farmers using GE crop technologies, conventional techniques, or organic production. In at least one case, a higher authority intervened to maintain peace. In May, Vermont’s Governor Jim Douglas vetoed a bill that would have made seed manufacturers liable for damages caused by GE seeds that drift into fields owned or occupied by a person with whom the seed manufacturer has not entered into a contract of sale, use, or license. The Governor called the measure unnecessary legislation that "dives into new legal territory that may only promote needless litigation that pits farmer against farmer and neighbor against neighbor."

A Summer Rerun in Congress
During the summer of 2003, Representative Dennis J. Kucinich (D-OH) introduced six bills aimed at the agbiotech industry. None emerged from House committee reviews.

In May, Kucinich introduced essentially the same six bills, which, he said, "are a common sense precaution to ensure genetically engineered foods do no harm." The ambitious legislation offers more than a few tweaks of current laws and regulations. "To ensure we can maximize benefits and minimize hazards," Kucinich said, "Congress must provide a comprehensive regulatory framework for all genetically engineered products."

HR 5266, "The Genetically Engineered Crop and Animal Farmer Protection Act," would initiate changes in federal agencies and in contracts between companies and farmers. The bill would amend the Federal Insecticide, Fungicide, and Rodenticide Act to direct the Administrator of the Environmental Protection Agency to establish the best achievable plan for preventing the development of resistance to Bacillus thuringiensis (Bt) toxin due to the introduction of GE plants that produce the toxin. The EPA would have to revoke Bt toxin registrations that fail to comply with the new plan and reduce the use of plant-incorporated Bt toxin if the agency determines that its use facilitates Bt-toxin resistance in pests, or if the agency’s review about Bt toxin resistance proves inconclusive.

The legislation would regulate contracts by requiring a company that sells any GE animal, plant, or seed for use in the United States to provide the purchaser with written notice of possible legal and environmental risks of the article’s use. The disclosure would not relieve the company from liability, and receipt of the notice by the purchaser would not create any liability for the purchaser. HR 5266 would also allow farmers to save seeds for future crop planting and prohibit the use of genetic engineering to generate plants that produce infertile seeds or seeds rendered infertile by the application of a chemical.

A moratorium on GE crops that produce pharmaceuticals or industrial chemicals would be initiated by HR 5267, the "Genetically Engineered Pharmaceutical and Industrial Crop Safety Act." The Act directs the Department of Agriculture to establish a tracking system to regulate the growing, handling, transportation, and disposal of all pharmaceutical and industrial crops and their byproducts to prevent contamination. Until the final regulations and tracking system have taken effect, the cultivation of a pharmaceutical crop or industrial chemical crop would be prohibited. The Act would also prohibit the cultivation of a pharmaceutical crop or industrial crop in an open air environment, and the production of such a crop from a plant commonly used for human food or domestic animal feed.

HR 5268, the "Genetically Engineered Food Safety Act," would amend the Federal Food, Drug, and Cosmetic Act to include GE food in the definition of "food additive." Kucinich said that this will create a more strenuous safety review process. The Act also details requirements that a company must meet in its petition to the Secretary of Health and Human Services for a regulation prescribing the conditions of safe use of a particular GE food additive. The Act authorizes civil actions against a company alleged to have violated provisions regulating GE food additives.

The labeling of GE food, an issue that has generated friction between the U.S. and European Union, may become a reality with HR 5269, the "Genetically Engineered Food Right to Know Act." The legislation would amend the Federal Food, Drug, and Cosmetic Act, the Federal Meat Inspection Act, and the Poultry Products Inspection Act to require that food that contains a GE material, or that is produced with a GE material, be labeled accordingly. The FDA would be required to periodically test products to ensure compliance.

HR 5270, the "Real Solutions to World Hunger Act," would restrict GE exports to GE products approved in the U.S. and approved by the importing nation. The bill would also create an international research fund for sustainable agricultural research paid by a fund supported from an income tax on companies engaged in genetic engineering.

The "Genetically Engineered Organism Liability Act," HR 5271, would place all liability resulting from an environmental release of a GE organism upon the biotech company that created the GE organism. Farmers would be granted indemnification to protect them from such liability, while biotech companies would not be allowed to transfer liability from the company.

Like their predecessors, the six bills sit in various House committees for review.

Selected Sources

Bay, A (2006) Proposed legislation would void county biotech seed bans. Capital Press (July 2006). Available: http://www.capitalpress.info.

Pew Initiative on Food and Biotechnology (2006) State Legislative Activity Related to Agricultural Biotechnology in 2005 (June 2006). Available: http://pewagbiotech.org.

Rathke L (2006) Governor vetoes GE seed liability bill. The Associated Press (May 16, 2006). Available: http://www.ruralvermont.org.

Speech of Hon. Dennis J. Kucinich of Ohio in the House of Representatives. Congressional Record E687-E688 (May 2, 2006).

 

Phill Jones
BiotechWriter.com
PhillJones@nasw.org


DOES THE ERA PROCESS WORK FOR GE CROPS?
Jeff Wolt and Saharah Moon Chapotin

This year marks the unofficial 25th anniversary of the ecological risk assessment (ERA) process as practiced within the federal government. The ERA process adheres to the paradigm of risk assessment as originally outlined in the NRC Redbook (NRC, 1983) with specific application to ecological risks. ERA has been defined as "the process that evaluates the likelihood that adverse ecological effects may occur or are occurring as a result of exposure to one or more stressors [or actions]" and is elaborated in both framework and guidance documents (USEPA, 1992; 1998). The ERA uses a ‘framework approach’ that presents a logical scheme for organizing complex information describing environmental exposure scenarios and effects to ecological entities of concern. The goals of an ERA framework are to

− develop a unified conceptual approach to environmental assessment;

− facilitate cooperation and collaboration between assessment-related disciplines;

− increase transparency of risk assessments to users (risk managers);

− provide standardized tools and techniques; and

− dispel the perception that ecological risk assessment is impossible (Barnthouse, 2006).

A key to success in applying ERA to a wide variety of technologies has been recognition that the ERA is a process – that is, a particular course of action intended to achieve a result (a procedure). ERA is not a technique, which is a specific approach to performing the assessment.

The timeline for evolution of the regulatory process for safety assessment for GE crops roughly parallels that for the ERA process. Current practice under the Coordinated Framework reflects the spirit of the ERA process but differs in some respects due to the nature of the technology involved. In applying ERA to GE crops, it is necessary to understand that the overall process of risk assessment does not start with the ERA. Rather the ERA relies on a body of precursor information that establishes with reasonable certainty that, other than for the expression of the trait of interest, the GE crop is equivalent to non-transformed comparators (see for example EuropaBio, 2003, 2004). Once equivalence has been established on the basis of the GE crop characterization, the ERA can proceed with emphasis on stressor-mediated effects, where the potential stressor (that is, the agent capable of causing harm) is the expressed trait, for instance a Bt protein conferring insect resistance to a crop. Thus, the general philosophy toward the ERA for GE crops:

− entails weight-of-evidence based on comprehensive evaluation of data;

− proceeds from general understanding to specific entities of concern;

− supports findings with quantitative data and analyses to the fullest extent possible;

− provides risk-based findings that focus on harm that may be manifested at environmentally relevant exposures; and

− seeks a determination of reasonable certainty of no harm to the environment or ecological entities in that environment.

It has been argued that even with these considerations – and 10 years of experience in safely growing GE crops – the ERA paradigm is inadequate to address long term ecological concerns arising from wide scale commercial production of GE crops (see for example, NRC, 2002). While the ERA process as it is applied to GE crops is consistent with the overall ERA framework, complexities exist due to the relatively recent nature of biotechnology and the fact that biological information is not fully quantifiable. Furthermore, GE crop risk assessments simultaneously consider the effect of stressors on individuals and populations as well as the effect of deployment on populations and communities. This leads to confusion by some with respect to how evaluation of stressor-mediated effects (the core consideration of ERA) can address uncertainties regarding effects at an ecological scale. In applying the ERA framework to GE crops, the stressor is recognized as the expressed product that elicits harm (for example, an insecticidally active Bt toxin). A relevant action is deployment of an event expressing the Bt toxin within a given region. Emphasis in the GE crop ERA should be given to the stressor-mediated effects from direct exposure, as the demonstration of reasonable certainty of no harm from direct exposure to the stressor provides reasonable certainty that secondary exposures arising from the action of deployment will not be ecologically relevant.

Considerable recent emphasis has been given to conceptual descriptions of how ERA can be applied to GE crops (Dutton et al., 2003; Wilkinson et al., 2003; Hill, 2005; Romeis et al., 2006a). In particular, there are several recent published examples of the application of ERA to concerns regarding GE crop effects to nontarget arthropods (NTA). Romeis et al. (2006b) have described the evaluation of insect resistant (IR) crop risks to biocontrol agents with a focus on beneficial insects whose exposure to plant toxins is indirect and involves tritrophic interactions. Wolt et al. (2003, 2005) and Peterson et al. (2006) have focused on IR crops expressing lepidopteran active toxins in demonstrating how laboratory-derived toxicity studies in conjunction with simple exposure estimates can address the likelihood of adverse consequence from inadvertent pollen consumption. In addition to these studies focused on risks to non-target arthropods, the broad methodology of ERA is generally applicable to questions of gene flow and weediness as recently described by Raybould and Cooper (2005).

These various studies demonstrate how the ERA for GE crops relies on a tiered process of both testing and subsequent assessment. This process proceeds from well-controlled, focused, laboratory studies conducted under very conservative assumptions regarding exposure potential, to less certain field studies and monitoring that seek the manifestation of hazard under real world conditions. Because controlled laboratory studies are conservative indications of likelihood for effect to be manifested under real world conditions (that is, of risk), the majority of GE crop ERAs conducted to date have relied on laboratory studies. In cases where confirmatory field studies and monitoring were conducted, laboratory study findings have proven adequate to determine that there is reasonable certainty of no harm associated with environmental release.

Summary
Regulatory experience with genetically engineered organisms (GEOs) in the United States provides a positive record of successful evaluations and subsequent safe use of the commercial products. This argues for a further streamlining of the process for regulatory approvals with a focus on the ecological risk assessment (ERA) framework as an objective science-based process for identification of risks that may adversely impact human health and the environment. To date, the ERA framework as applied to GEOs has been sufficiently flexible to deal with concerns surrounding biotechnology. The use of common data elements to address case-specific problems serves as important precursor information that directs the ERA toward risk assessment for consequential concerns.

Regardless of the particular environmental or ecological concern that may be addressed, the ERA is a science-based process that evaluates exposure and effect (the consequence of the exposure in terms of likelihood of harm). Focusing on the consequences of environmental release of GE crops rather than concerns regarding GE crop deployment provides an objective means to use science-based information for regulatory and public policy decisions.

References

Barnthouse L. (2006) Strengths of the Ecological Risk Assessment Process for Use in Decision Making, Presentation to the USEPA Science Advisory Board Ecological Processes and Effects Committee (EPEC) Workshop on Ecological Risk Assessment – An Evaluation of the State-of-the-Practice, 7- 8 Feb 2006, Washington, D.C.

Dutton A, Romeis J and Bigler F (2003) Assessing the risks of insect resistant transgenic plants on entomophagous arthropods: Bt-maize expressing Cry1Ab as a case study, BioControl 48, 611

EuropaBio (2003) Safety assessment of GM crops, Document 1.1 Substantial equivalence – maize, European Association for Bioindustries, (http://www.europabio.org/relatedinfo/CP11.pdf)

EuropaBio (2004) Assessing the safety of genetically modified plants for non-target organisms, European Association for Bioindustries, (http://www.europabio.org/relatedinfo/Poster_IOBC_Montpellier_2004.ppt)

Hill RA (2005) Conceptualizing risk assessment methodology for genetically modified organisms, Environ. Biosafety Res. 4, 67-70

[NRC] National Research Council (1983) Risk Assessment in the Federal Government: Understanding the Process, National Academy Press, Washington DC.

[NRC] National Research Council (2002) Environmental Effects of Transgenic Plants: The Scope an Adequacy of Regulation, National Academy Press, Washington DC.

Peterson RK, Meyer SJ, Wolf AT, Wolt JD, and Davis, PM (2006) Genetically engineered plants, endangered species, and risk: A temporal and spatial exposure assessment for karner blue butterfly and Bt maize pollen, Risk Analysis 26, 845-858

Raybould A and Cooper I (2005) Tiered tests to assess the environmental risk of fitness changes in hybrids between transgenic crops and wild relatives: the example of virus resistant Brassica napus, Environ. Biosafety Res. 4, 127-140

Romeis J et al. (2006) Moving through the tiered and methodological framework for non-target arthropod risk assessment of transgenic insecticidal crops, Proc. 9th International Symposium on Biosafety of Genetically Modified Organisms (ISBGMO), 24-29 Sep 2006, Jeju Island, South Korea, (submitted)

Romeis J, Meissle M, and Bigler F (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology 24, 63 - 71

[USEPA] United States environmental Protection Agency (1992) Framework for Ecological Risk Assessment, Risk Assessment Forum, EPA/630/R-92/001

[USEPA] United States environmental Protection Agency (1998) Guidelines for Ecological Risk Assessment, Risk Assessment Forum, EPA/630/R-95/002F.

Wilkinson MJ, Sweet J, and Poppy GM (2003) Risk assessment of GM plants: Avoiding gridlock? Trends Plant Sci. 8, (5), 208-212

Wolt JD, Conlan CA, and Majima K (2005) An ecological risk assessment of Cry1F maize pollen impact to pale grass blue butterfly. Environmental Biosafety Research 4, 243-251

Wolt, JD et al., (2003) A screening level approach for non-target insect risk assessment: Transgenic Bt corn pollen and the monarch butterfly (Lepidoptera: Danaiidae), Environ. Entomol. 32, 237-246

 

 

Jeff Wolt and Saharah Moon Chapotin
Biosafety Institute for Genetically Modified Agricultural Products (BIGMAP)
Iowa State University, Ames, IA
jdwolt@iastate.edu



MYCOTOXIN REDUCTION IN BT CORN: Potential Economic, Health, and Regulatory Impacts
Felicia Wu

Introduction
Transgenic Bt corn contains a gene from the soil bacterium Bacillus thuringiensis that encodes for a crystal (Cry) protein that is toxic to common lepidopteran corn pests. Because of reduced pest damage, one indirect benefit of Bt corn is lower levels of mycotoxin contamination. Foodborne mycotoxins are secondary metabolites of fungi that can be toxic, carcinogenic, or both to humans and animals. Insect damage is one factor that predisposes corn to mycotoxin contamination, because insect herbivory creates kernel wounds that encourage fungal colonization, and insects themselves serve as vectors of fungal spores (Munkvold and Hellmich 1999). Thus, any method that reduces insect damage in corn also reduces risk of fungal contamination. Indeed, in a variety of field studies, Bt corn has been shown to have significantly lower levels of common mycotoxins. In less developed countries, the mycotoxin reduction that Bt crops can provide could have important economic as well as health impacts. Thus, it is an important phenomenon to consider when developing regulatory policies on Bt crops.

Mycotoxins in corn: fumonisin and aflatoxin
Two of the most important mycotoxins in corn are fumonisins and aflatoxins. Fumonisins are produced by the fungi Fusarium verticillioides and Fusarium proliferatum. Consumption of fumonisin has been associated with elevated human esophageal cancer incidence in various parts of Africa, Central America, and Asia (Marasas et al. 2004) and among the black population in Charleston, South Carolina (Sydenham et al. 1991). Because fumonisin B1 reduces uptake of folate in different cell lines, fumonisin consumption has been implicated in neural tube defects in human babies (Marasas et al. 2004). Fumonisins can be highly toxic to animals, causing diseases such as equine leukoencephalomalacia (ELEM) in horses and porcine pulmonary edema (PPE) in swine (Ross et al. 1992).

Aflatoxins are produced by the fungi Aspergillus flavus and Aspergillus parasiticus, and are the most potent chemical liver carcinogens known. Acute aflatoxicosis, characterized by hemorrhage, acute liver damage, and possibly death, can result from extremely high doses of aflatoxin. For people who are infected with hepatitis B and C (common in East Asia and sub-Saharan Africa), aflatoxin consumption raises more than tenfold the risk of liver cancer compared with either exposure alone. Aflatoxin consumption is also associated with stunting in children and immune system disorders (Turner et al. 2003). In poultry, aflatoxin consumption results in liver damage, decreased egg production, inferior egg-shell quality, inferior carcass quality, and increased susceptibility to disease (Wyatt 1991). In cattle, the primary symptoms are reduced weight gain, liver and kidney damage, and reduced milk production (Keyl 1978).

Currently existing fumonisin and aflatoxin standards
Many nations have established foodborne mycotoxin regulations in food and animal feed. In the U.S., the Food and Drug Administration (FDA) has set industry guidelines for levels of fumonisin acceptable in human food and animal feed. The most stringent of these standards applies to degermed dry-milled corn products for human food, with a recommended total fumonisin maximum level of 2 mg/kg. Few other nations currently have fumonisin standards for food.

The presence of aflatoxins in foods is restricted in the U.S. to the minimum levels practically attainable by modern processing techniques. The most stringent of these FDA standards for corn is 20 μg/kg total aflatoxin. Many other nations have established maximum tolerated levels of aflatoxin in food and feed. Notably, the European Commission has set a total aflatoxin standard of 4 parts per billion (µg/kg) in food, the strictest standard worldwide.

Wu (2004) calculated that corn export market losses to the U.S., China, Argentina, and Africa could total in the hundreds of millions of dollars ($US) annually if strict mycotoxin standards, such as those of the European Union, were adopted more broadly worldwide. While the U.S. as top corn exporter would experience the greatest loss, China, Argentina, and Africa’s losses would represent a much larger proportion of their total export market. As mycotoxin standards are expected to become stricter over time, it is important to consider effective methods to reduce mycotoxins in food crops.

Evidence for Bt corn reducing mycotoxin contamination
Where insect pests are present, Bt corn has been shown to have lower levels of certain mycotoxins than non-Bt isolines. In the Corn Belt region of the United States, when insect damage from the European corn borer (ECB) or Southwestern corn borer (SWCB) is high, fumonisin concentrations are significantly lower in Bt corn compared with their near-isogenic, non-transgenic counterparts (Munkvold and Hellmich 1999). In France, Italy, Turkey, and Argentina, Bt corn has been shown in field trials to have significantly lower fumonisin levels than non-Bt isolines (Hammond et al. 2003).

Compared with fumonisin, insect pest damage is less strongly correlated with aflatoxin concentrations in corn.  The insects that are controlled by Bt corn are not as important in predisposing plants to infection by A. flavus as they are for F. verticillioides and F. graminearum; and A. flavus can infect corn not just through kernel wounds caused by insects, but through the silks.  Hence, field tests of aflatoxin reduction in Bt corn show a mixed record.  Williams et al. (2002) found that in controlled studies involving spraying young ears with A. flavus inoculum, Bt corn had significantly lower levels of aflatoxin than non-Bt corn.  Other studies show no significant effect of Bt corn, or mixed results (Buntin et al. 2001, Odvody et al. 2000). 

Economic impacts of Bt corn in mycotoxin reduction
Wu et al. (2004) developed a model to assess the economic benefits in the United States due to Bt corn’s reduction of mycotoxins. Three classes of economic impacts from mycotoxins are identified: market effects, animal health, and human health. High quality corn (i.e., low levels of mycotoxin) can be sold as human-food-grade corn at the highest market price. Corn contaminated with levels of mycotoxins between the highest-permitted levels of food and feed can be sold for animal feed at a lower price, and corn with high levels of mycotoxins is either sold for non-food-non-feed uses at an even lower price or rejected outright. The proportions of the total crop that are rejected at each of these levels depend on the national or international standards for mycotoxins in food and feed. Animal health losses are estimated by total number of susceptible animals multiplied by their market value. No human health benefits of Bt corn’s mycotoxin reduction were estimated because it is exceedingly rare for any human health impacts to occur from mycotoxin consumption in the United States. To then estimate what the impact of Bt corn planting would be, it was assumed that where ECB or SWCB were the predominant pests, Bt corn would reduce fumonisin to levels safe for human consumption 80-95 percent of the time, and would reduce aflatoxin to safe levels 50 percent of the time.

It was estimated that in the U.S., the average total annual loss due to fumonisins in corn is about $40 million (range: $14M to $88M; values in parentheses represent the 95% confidence level, Table 1). The annual market loss in the U.S. from corn rejected either for food or for feed makes up most of this loss: roughly $39 million ($14M to $86M). Of this amount, about $38 million of the estimated losses are through corn rejected for food, and slightly less than $1 million of the losses are through corn rejected for feed.

Using the assumption that Bt corn contains fumonisins at or below the FDA standard for human consumption 80-95% of the time, the savings to U.S. farmers from increased market acceptance is estimated at $8.8 million annually ($2.3M to $31M). The total value of animal mortality from fumonisin consumption is relatively small in the U.S. because in most years fumonisin levels are sufficiently low that few, if any, animals are affected in most regions of the U.S. The estimated annual loss from fatal fumonisin-induced ELEM in horses is $270 thousand ($51 thousand to $2 million). In swine, the annual expected losses from fumonisin-induced PPE are on the order of several tens of thousands of dollars. These deaths occur on farms that grow their own corn rather than buying commercial feed, which presumably has safe fumonisin levels. The benefit of planting Bt corn in preventing swine and horse mortality is estimated to be $67 thousand annually ($13 thousand to $500 thousand).

It was estimated that in the U.S., the total annual loss due to aflatoxin in corn is about $163 million ($73 million to $332 million). The annual market loss through corn rejected for food is about $31 million ($10M to $54M), while the loss through corn rejected for feed and through livestock losses is estimated at $132 million ($14M to $298M). Bt corn would reduce aflatoxin in cases where insect damage from Bt-sensitive insects was the main determinant of aflatoxin development. Given the current level of Bt corn planting in such regions at about 17% (USDA 2004), and the assumption that Bt corn is partly effective in reducing aflatoxin only in Texas / Southeastern U.S. where 80% of the aflatoxin contamination problems occur, the upper limit of the current benefit is $14 million ($5.0M to $22M).

Table 1 summarizes the economic losses due to fumonisin and aflatoxin in corn in the U.S., and the benefits that Bt corn currently provides in terms of reducing mycotoxin contamination.

While these calculations are relevant specifically to the United States, the model is easily adapted to other nations that are Bt corn adopters, or potential Bt corn adopters. Adjustments must be made as to rates of adoption, types of pests that are common in the different regions, and acceptable levels of fumonisin and aflatoxin for food and feed as set by the government.

Discussion
Mycotoxins in corn result in multiple adverse health effects as well as dramatic market-related losses. One potential technology to control mycotoxins is cultivation of Bt corn. Where Bt corn is planted, depending on the severity of other impacts such as weather conditions, it often has significantly reduced fumonisin and aflatoxin when pest infestation would otherwise cause high levels of these mycotoxins.

In the United States, where roughly a quarter of total field corn acreage is planted with Bt corn, the annual benefits that Bt corn provides in terms of lower fumonisin and aflatoxin contamination are estimated at about $23 million. It is likely that animal and human health benefits of Bt corn would be more prominent than market gains in areas such as Latin America and sub-Saharan Africa, and the northern regions of China where corn is a staple in animal and human diets and mostly exchanged locally.

In the future, as mycotoxin standards may become stricter and thereby harder to meet, mycotoxin-control technologies such as Bt corn may grow in popularity among corn-exporting nations worldwide.

References

Buntin GD et al. (2001) Evaluation of Yieldgard transgenic resistance for control of Fall Armyworm and Corn Earworm (Lepidoptera: Noctuidae) on corn. Florida Entomologist 84(1), 37-42

Hammond B et al. (2003) Reduction of fumonisin mycotoxins in Bt corn. The Toxicologist 72(S-1), 1217

Keyl AC (1978) Aflatoxicosis in cattle. In: Mycotoxic Fungi, Mycotoxins, Mycotoxicoses, vol. 2; Wyllie TD, Morehouse LG, (Eds), Marcel Dekker: New York, pp. 9-27

Marasas WFO et al. (2004) Fumonisins disrupt sphingolipid metabolism, folate transport, and neural tube development in embryo culture and in vivo: A potential risk factor for human neural tube defects among populations consuming fumonisin-contaminated maize. J Nutrition 134, 711-716

Munkvold GP & Hellmich RL (1999) Comparison of fumonisin concentrations in kernels of transgenic Bt maize hybrids and nontransgenic hybrids." Plant Disease 83(2), 130-138

Odvody GN et al. "Aflatoxin and insect response of near-isogenic Bt and non-Bt commercial corn hybrids in south Texas" in Proceedings of the 2000 Aflatoxin/Fumonisin Workshop, J.F. Robens, ed. USDA Agricultural Research Service, Beltsville, MD, 2000

Ross PF et al. (1992) A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates. Mycopathologia 17, 109-114

Sydenham EW et al. (1991) Fumonisin contamination of commercial corn-based human foodstuffs." Journal of Agricultural Food Chemistry 39, 2014-2018

Turner PC et al. (2003) Modification of immune function through exposure to dietary aflatoxin in Gambian children. Environmental Health Perspectives 111, 217-20

Williams WP et al. (2002) Aflatoxin accumulation in conventional and transgenic corn hybrids infested with Southwestern Corn Borer (Lepidoptera: Crambidae)." Journal of Agricultural and Urban Entomology 19(4), 227-236

Wu F (2004) Mycotoxin risk assessment for the purpose of setting international regulatory standards. Environmental Science & Technology 38(15), 4049-4055

Wu F, Miller JD, & Casman EA (2004) Bt corn and mycotoxin reduction: An economic perspective. Journal of Toxicology, Toxin Reviews 23(2-3), 397-424

Wyatt RD (1991) Poultry. In Mycotoxins and animal foods; Smith, J.E; Henderson, R.S., Eds.; CRC Press: Boca Raton, FL., pp 553-606

Felicia Wu
Assistant Professor of Environmental and Occupational Health
University of Pittsburgh
Pittsburgh, PA
fwu@eoh.pitt.edu


INDUSTRIAL BIOTECH WILL ACCOUNT FOR 10% OF CHEMICAL INDUSTRY BY 2010

Industrial biotechnology will account for 10 percent of sales within the chemical industry by 2010, accounting for $125 billion in value. That’s according to a report released by McKinsey & Company, a global business consulting firm, at the third annual World Congress on Industrial Biotechnology and Bioprocessing held mid July in Toronto.

McKinsey & Company partner Jens Riese said the firm has 90 percent confidence in this projected growth, based on the current value of industrial biotechnology. Already as of 2005, industrial biotechnology – counting products made from biobased feedstocks or through fermentation or enzymatic conversion – accounts for 7 percent of sales and $77 billion in value within the chemical sector.

Much of the projected growth in adoption of industrial biotechnology is attributable to biofuels – ethanol and biodiesel – as production is rapidly increasing to meet demand driven by government mandates.

However, Riese stressed, to meet future demand and maintain growth, biofuel production will have to adopt biotechnology processes that make use of broader feedstocks, including cellulose biomass. "To meet demand just from mandated usage, we need to do something different. The key to expanding biofuel production is tapping into new agricultural feedstocks," Riese said.

Biotechnology May Drive Down the Cost of Biofuels
A panel of executives representing BP, DuPont, and Chevron offered their perspectives on the intersection of the energy and chemical industries with the industrial biotechnology and life sciences sectors, at the recent World Congress on Industrial Biotechnology and Bioprocessing. The executives highlighted both the need for biofuels and other forms of energy and the opportunities that their respective companies are pursuing through industrial biotechnology.

Justin Adams, Director, Long Term Technology Strategy with BP, began the discussion by saying that the key drivers of the energy future will be supply security and environmental constraints. He noted that biofuels can help to meet these challenges, saying, "Biotechnology holds the key to driving down the costs of biofuel production" throughout the value chain, including the development of new feedstocks, novel enzymes, and fermentation technology. "What chemistry did in the 20th century, biology will do in the 21st," Adams said.

Bill Provine, Research Manager with DuPont discussed his company’s strategy for using industrial biotechnology throughout the value chain, from specialized agricultural feedstocks to biotech enzyme and fermentation processes that produce biobutanol, a new type of biofuel. Richard Zalesky, Vice President of Biofuels & Hydrogen Business Unit, Chevron Technology Ventures, outlined a partnership with the state of California and Pacific Ethanol to study the use of E85 in state-owned vehicles as well as a collaboration with The Georgia Institute of Technology aimed at making cellulosic biofuels, biodiesel and hydrogen viable transportation fuels.




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