Transparency and documentation of the decision process are at the core of a credible risk assessment and, in addition, are essential in the presentation of a weight of evidence (WoE)-based approach. Lack of confidence in the risk assessment process (as the basis for a risk management decision), beginning with evaluation of raw data and continuing through the risk decision process, is largely because of issues surrounding transparency. There is a critical need to implement greater transparency throughout the risk assessment process, and although doing so will not guarantee the correctness of the risk assessment or that all risk assessors come up with the same conclusions, it will provide essential information on how a particular conclusion or decision was made, thereby increasing confidence in the conclusions. Recognizing this issue, the International Life Sciences Institute Health and Environmental Sciences Institute convened a multisector committee tasked with discussing this issue and examining existing guidance and recommendations related to transparency in risk assessment. The committee concluded that transparency is inextricably linked to credibility: credibility of the data, credibility of the risk assessment process, and credibility of the resulting decision making. To increase this credibility, existing guidance concerning criteria elements of transparency related to the risk assessment process must be more widely disseminated and applied, and raw data for studies used in human health and environmental risk assessment must be more widely available. Finally, the decision-making process in risk management must be better documented and a guidance framework established for both the process itself and its communication to the public.
Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.
Protoporphyrinogen IX oxidase (protox) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the penultimate step of heme and chlorophyll biosynthesis in animals and plants. Protox is the target of light-dependent peroxidizing herbicides and is inhibited at nanomolar levels by several chemical classes including tetrahydrophthalimides (discussed below) and diphenyl ethers (e.g., acifluorfen) usually with little selectivity between the mammalian and plant enzymes. The herbicide binding site is examined here with a photoaffinity radioligand optimized on the basis of structure-activity relationships. A radiosynthetic procedure is described for this new herbicidal probe, N-(5-azido-4-chloro-2-fluorophenyl)-3,4,5, 6-[3H]tetrahydrophthalimide ([3H]AzTHP), resulting in high specific activity (2.6 TBq/mmol). Human protox expressed in Escherichia coli and purified by affinity chromatography is used with [3H]AzTHP to characterize the herbicide/substrate binding site. Specific binding of [3H]AzTHP to human protox is rapid, completely reversible in the absence of light with a Kd of 93 nM, and competitively inhibited by the 5-propargyloxy analogue and by acifluorfen, which are known to bind at the substrate (protoporphyrinogen) site. The Bmax establishes one [3H]AzTHP binding site per FAD. Diphenyleneiodonium, proposed to inhibit protox by interaction with the FAD cofactor, inhibits enzyme activity by 48% at 100 micro M without affecting [3H]AzTHP binding in the presence or absence of substrate, suggesting that the herbicide binding site may not be proximal to FAD. The first step has been taken in photoaffinity labeling the herbicide/substrate site with [3H]AzTHP resulting in apparent covalent derivatization of 13% of the herbicide binding site.
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