Drug-induced liver disease (DILD) is a common cause of life-threatening, acute liver failure and is a major reason drugs are removed from clinical development and widespread use. Because of its idiosyncratic nature, accurate prediction of which new drugs will cause DILD and who will be at risk for the development of this disease is difficult. Likewise, the pathogenesis of DILD is complex and appears to involve the formation of reactive drug metabolites that affect critical biochemical functions or elicit an immune response. Recent studies, however, have demonstrated that a deficiency of key protective factors in the liver – such as anti-inflammatory cytokines and others – can lead to increased tissue susceptibility to DILD, often through unchecked protoxicant activities. In this regard, we tested the hypothesis that IL-13, an anti-inflammatory cytokine, is another such factor. Administration of an IL-13 neutralizing antibody (NAb) two hours before acetaminophen (APAP) treatment in wild-type (WT) mice attenuated serum IL-13 concentration and significantly exacerbated liver injury up to 24 hours after APAP administration. While the qualitative nature of the resulting centrilobular lesions was similar, morphometric analysis revealed that IL-13 NAb pretreatment significantly increased lesion area. Moreover, APAP administration to IL-13 knockout (KO) mice confirmed the protective role of IL-13 in APAP-induced liver injury. Serum TNF-? concentration was significantly elevated in APAP-treated IL-13 KO mice and in WT mice co-administered with IL-13 NAb and then APAP. In contrast, no differences were observed in protein adduct formation or detoxifying liver glutathione levels between APAP-treated IL-13 deficient and WT mice. Taken together, these results suggest that IL-13 is a critical hepatoprotective factor, modulating the expression of protoxicant mediators but not APAP metabolism. Further elucidation of the hepatoprotective role of IL-13 will be useful in better understanding the mechanism and in facilitating the prediction of DILD susceptibility, thereby preventing the removal of otherwise beneficial drugs from the market.

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Hepatotoxicity caused by drug-induced liver injury (DILI) is a dangerous and increasing problem. It has become the leading cause of acute liver failure in the United States, exceeding all other causes combined, including viral infections and alcoholic liver disease. It is also a principal reason for withdrawing approved drugs from the market. As toxicologists you work to prevent this by detecting liver injury in animals during the pre-clinical and non-clinical phases of new drug development. Yet those efforts sometimes fail. Let us consider this evening some of the possible reasons for this.

As an internist and hepatologist, I work with the species Homo sapiens, a much less standardized animal and one much more difficult to control than the species with which you work. Although laboratory animals are bred to be genetically as much alike as possible, and conditions of diet, drug administration, and environmental control are rigorously controlled in your studies, that cannot be so for humans. Our species is highly skilled at mixing and muddling its gene pool, is very resistant to being told what to do, what to eat, what drugs to take, what dietary supplements to use.

It is a problem of the numbers that causes much difficulty. Serious DILI is relatively uncommon or rare, thanks to your diligent work in excluding the really toxic drugs. But it still occurs in some people, and when hundreds of thousands or millions are exposed to a drug, even a few hundred cases of acute liver failure may be unacceptable. Another problem of the numbers is that the rare, idiosyncratically susceptible person may not be found in a controlled clinical study even of several hundred patients (it takes about three times the inverse of the incidence to have a 95% chance of finding at least one such person, i.e., if the true incidence of the problem is 1 per 1,000 then 3,000 must be studied). Even more numerical problems arise when we consider how to detect the liver injury when it does happen. For rare events, a very sensitive test is not good enough, and extraordinarily specific tests are needed to avoid being inundated with false-positive results in those who do not have significant DILI.

Monitoring for detecting liver injury is usually not done as recommended, may be unable to prevent serious damage even if done, and false positive test results create excessive costs with little proved benefit. Even when liver injury is detected, too often not enough information is gathered or reported to make possible the exclusion of non-drug-related cause of the abnormalities seen, and only a small fraction of the actual cases are reported at all to our adverse event reporting system.

A drug-induced liver injury network of six medical centers has been established and funded by the Liver Section of the National Institute for Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health to begin to address these problems. We at the FDA are interacting and cooperating with them to facilitate this endeavor. We need both to sharpen our methods for detecting DILI, making accurate differential diagnosis and correct attribution of causality as indeed drug-induced, and then to find ways to fund and carry prospective safety studies to define true incidence of DILI in people exposed, identify risk factors that make certain individuals more susceptible, and focus on learning about the mechanisms by which injury occurs. We then can initiate rational risk management procedures, and demonstrate their value by showing quantitative reduction or elimination of the problems.

Learning Objectives: after hearing and seeing this presentation, listeners should be able to

1. state two reasons why the problem of post-marketing drug-induced liver injury has arisen;
2. list consequences of it for the FDA, PhRMA, the medical profession, and patients;
3. explain why DILI tends to be relatively rare but serious;
4. explain the difference between test sensitivity and specificity, and why the latter is important;
5. describe what information may be needed to reduce false positive attribution of DILI;
6. understand the need for and value of prospective, controlled safety studies.

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Dr. Lester M. Crawford, Deputy Commissioner of the FDA and the current Acting Commissioner, was the keynote speaker at the October 3 meeting of the Association of Government Toxicologists (AGT). Dr. Crawford addressed the AGT on "FDA's Toxicology Challenges Today and Tomorrow" at the Lister Hill Center, which is located on the campus of the National Institute of Health in Bethesda. His presentation was well received by the audience of AGT members and guests.

Before discussing toxicology, Dr. Crawford provided a brief overview of the several pressing issues before FDA, such as Dr. McClellan's Senate confirmation hearing and the Agency's recent hiring efforts. He mentioned that just like when the Agency was in its infancy, chemistry is the most common scientific discipline found among its new hires. He also quipped that "it only takes a little more training" to convert a chemist into a toxicologist. Dr. Crawford noted that the future of FDA depends on the 800 new hires that were brought aboard since the beginning of his tenure in February, 2002.

In the area of toxicology, he discussed such varied issues as acrylamide, dioxin, and dietary supplements such as ephedra. He also provided an insider's view and Agency update on antibiotic resistance and on the transmissible encephalopathies such as BSE and chronic wasting disease in elk and deer.

After Dr. Crawford's overview, he opened the meeting to a question and answer period, which resulted in a lively discussion with the AGT members and guests. The attendees expressed interest in the toxicology of dietary supplements, methyl mercury in seafood, and the potential impact of proteomics and geonomics on toxicology.

-by Ron Varsaci

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Dr. Harry Salem was the Society of Toxicology's 3^rd Congressional Science Fellow. The aim of this program is to increase the Society's visibility in Washington, and to support the use of scientific expertise in governmental decisions. The challenges for a scientist thrust among politicians, the initiatives taken, and the outcomes and impacts will be discussed. These will include placement with Congressman Jim Greenwood from the 8^th district in Pennsylvania, who is Chair of the Subcommittee on Oversight and Investigations of the House Energy and Commerce Committee. This Subcommittee has the broadest jurisdiction of any subcommittee in Congress. Among the governmental entities answerable to Greenwood's Subcommittee are the National Institutes of Health, the Federal Communications Commission, and the Environmental Protection Agency. Dr. Salem was tasked by Congressman Greenwood to address his environmental issues of concern, all of which are in the public's eye. These include arsenic in drinking water, oxygenates in gasoline, global warming, health tracking and bioterrorism. These issues, and the initiatives taken, as well as the interactions with the congressman on these, the impacts and status will be addressed.

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In vitro percutaneous absorption methods have been widely used for determining the absorption of compounds that are applied to skin. Safety evaluations of toxic chemicals frequently rely on in vitro absorption studies in human skin to help in the estimation of exposure. Animal absorption data must be used cautiously for estimating human absorption due to differences in the barrier properties of animal and human skin. In vitro absorption studies can also be used to measure skin metabolism if viable skin is used and if viability of skin is maintained in the test system. The in vitro nature of the diffusion cell allows for the isolation of the skin so that dermal metabolism can be differentiated from systemic metabolism. During this presentation we will review the skin absorption and metabolism of 2-nitro-p-phenylenediamine (hair dye), disperse blue 1 (hair dye), dihydroxyacetone (tanning agent) and a fluorescent laundry brightener.

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Fumonisins, mycotoxins derived from Fusarium verticillioides, are commonly found in corn and corn products. Fumonisins produce species-specific toxic responses, e.g., porcine pulmonary edema, equine leukoencephalomalacia, liver and kidney toxicity and carcinogenicity in rodents, and are suspected human carcinogens. Fumonisins are structurally related to sphingoid bases and inhibit ceramide synthase, an enzyme important in sphingolipid metabolism, thereby causing an accumulation of sphingoid bases in target tissues. The tissue and cell-selective responses to fumonisins include proliferation, necrosis as well as signal-mediated programmed cell death (apoptosis), but in some cases, fumonisins inhibit apoptosis. The acute hemopoietic effects of FB₁ were abrogated by pretreatment with anti-TNFalpha. In mice treated with FB₁ there was consistently an increased expression of TNFalpha in liver, less so in kidney, but not in spleen, thus suggesting that the source of TNFalpha was not peripheral macrophages. Hepatotoxic responses to FB₁ were reduced in TNF receptor-1 or 2-knockout mice. Taken together, the results strongly suggest that TNFalpha is involved in FB₁-induced apoptotic response, and that the TNFalpha pathway is somehow related to the accumulation of sphingoid bases. Modulation of TNFalpha is however not dependent on sphingoid metabolism. TNFalpha is also important in repair of hepatic damage; mice lacking the pathways for this cytokine were more sensitive to the toxic effects of FB₁. Exposure of mice to FB₁ results in modulation of a variety of genes involved in cell signaling, cycling, and regulation of other molecules.

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The goal of our team is to be able to use in silico approaches to accurately predict the toxicity in animals and humans of any chemical submitted to FDA at any toxicological endpoint normally examined by FDA during its regulatory considerations.

Based on the success we are having in producing high-quality computational toxicology modules, I made a "prediction" to Jonathan C. Peck, Vice President, Institute for Alternative Futures, at the recent FDA Science Forum³ that FDA would be able to establish the safety of a drug based solely on in silico data by 2007. The drug so approved will be one that is structurally very similar to ones that have already been adequately tested by traditional means, and because of this, with the use of high quality QSAR software, no in vitro tests, animal assays, or human clinical trials will be necessary⁴.

One of the reasons I enjoy attending and speaking at AGT meetings is that "larger issues," even "philosophical considerations" are often discussed instead of the "what I did in my lab last week" that is the usual fare at scientific meetings. During my AGT talk I will explain in more detail what our group does and the success we are having, but then I will go on and discuss what I think computational toxicology will enable FDA and other regulatory agencies to do. I will say that I believe: 1) That the computer power necessary for successfully predicting safety in silico for most FDA-regulated substances has already been achieved; 2) That the software needed to predict accurately the toxicological properties of most FDA-regulated substances in animals and humans will be in hand in the next three years; but 3) That the most difficult to surpass barrier to the actual implementation of FDA approval based on computer-based information is resistance to new approaches by "practitioners in the field"; including, perhaps, some of the people who will be in the very room in which I will be giving my talk!

If you can come to this meeting, I hope you will be entertained and will feel free to join in the discussion.

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¹This title is adapted from a statement that was presented by Jonathan C. Peck, Vice President, Institute for Alternative Futures, at the USFDA Science Forum, February 15, 2001.

²All opinions expressed in this summary and in my talk are mine and are not necessarily shared by USFDA.

³At which he made a presentation on Learning In Silico: 21^st Century Product Development.

⁴Computers have no imagination; they can only predict future behavior if they have a solid database of information of past research and testing on which to base their estimates. Because of this, there will always be a need for the biological testing of molecular entities that are truly novel.

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Despite the U.S. ratification of the Chemical Weapons Convention in 1997, in which the U.S. agreed to demilitarize and destroy our stockpile of chemical warfare agents, there is still potential for human exposure to these compounds and thus a need for health-based standards. Potential for worker and general population exposures is relatively small, but involves many different scenarios - from single, acute exposures in the event of an accident or terrorist attack, to potential long-term low concentration exposures associated with workplace environments, disposal facility emissions, or residuals in soil from past disposal practices. While the U.S. Army Office of the Surgeon General is the lead agency responsible for obtaining and evaluating toxicity data for these military-unique compounds, over the past several years the Army has looked to non-military partnerships to establish health standards based on broader scientific expertise, standardized risk assessment practices, and improved public and regulatory acceptance. This presentation will provide an overview of the toxicity and physicochemical characteristics of primary chemical compounds considered "warfare agents," as well as a status report of various initiatives to establish occupational and environmental health standards for these compounds.

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Dr. Stack will be discussing the recently-released National Research Council report, Scientific Frontiers in Developmental Toxicology and Risk Assessment. This report reviews advances made during the last 10-15 years in fields such as developmental biology, molecular biology, and genetics. The report describes a novel approach for how these advances might be used in combination with existing developmental toxicology methodologies to further the understanding of mechanisms of developmental toxicity, to improve the assessment of chemicals for their ability to cause developmental toxicity, and to improve risk assessment for developmental defects. For example, based on the recent advances, even the smallest, simplest laboratory animals such as the fruit fly, roundworm, and zebrafish might be able to serve as developmental toxicological models for human biological systems. Use of such organisms might allow for rapid and inexpensive testing of large numbers of chemicals for their potential to cause developmental toxicity; presently, there are little or no developmental toxicity data available for the majority of natural and manufactured chemicals in use. This new approach to developmental toxicology and risk assessment will require simultaneous research on several fronts by experts from multiple scientific disciplines, including developmental toxicologists, developmental biologists, geneticists, epidemiologists, and biostatisticians.

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It is increasingly becoming clear that a large number of environmental contaminants can alter the immune functions thereby leading to increased susceptibility to infections, autoimmunity, allergies and cancer. Our lab is primarily interested in identifying the mechanisms by which such contaminants including dioxin (TCDD) alter the immune functions. Studies from our lab have demonstrated that TCDD induces thymic atrophy and immune dysfunction by triggering apoptosis. Furthermore, we have shown that TCDD increases the expression of a critical gene, Fas ligand (FasL) that induces apoptosis following interaction with Fas receptor. Because Fas is expressed not only on immune cells but also on other tissues including testes, liver, intestine and heart, it is likely that TCDD-induced upregulation of FasL may lead to generalized toxicity. To confirm our hypothesis we have used mice that are deficient in Fas and FasL and have shown that they are more resistant to the toxicity caused by TCDD. Also, caspase inhibitors that block apoptosis were found to neutralize TCDD-induced immunotoxicity. TCDD was shown to promote FasL based activation induced cell death in peripheral T cells following antigen stimulation. TCDD is known to bind to the Ah receptor and activate a plethora of genes. Thus, using gene microarray technology, we have screened hundreds of genes simultaneously involved in the regulation of apoptosis and cell proliferation. Using this technology, we have shown that TCDD induces the expression of a large number of genes involved in apoptosis and cytokine production. These studies have also shown that gene microarray technology can be used as a sensitive assay for the detection of biomarkers for immunotoxicity.

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The molecular biology revolution has facilitated rapid advances in our understanding of the molecular basis of cellular structure, function, and defense. An appreciation of the evolution of inducible defense systems and the cellular responses to molecular damage, combined with the availability of powerful genomic, proteomic, and informatics technologies, has created an unprecedented opportunity to identify biomarkers of cell and tissue damage. Additionally, improved imaging technologies make possible noninvasive monitoring of molecular biomarkers in animal and human studies. The potential impact of these advances on toxicological practice will be discussed.

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Sponsors of Study

• U.S. Environmental Protection Agency
• U.S. Department of Interior
• Centers for Disease Control and Prevention

Committee on Hormonally Active Agents in the Environment

• Ernst Knobil, Chair, University of Texas, Houston Medical School
• Howard A. Bern, University of California, Berkeley
• Joanna Burger, Rutgers University
• D. Michael Fry, University of California, Davis
• John P. Giesy, Michigan State University
• Jack Gorski, University of Wisconsin
• Charles J. Grossman, Xavier University
• Louis J. Guillette, Jr., University of Florida
• Barbara S. Hulka, University of North Carolina
• James C. Lamb, Jellinek, Schwarz, & Connolly, Inc.
• Leslie A. Real, Emory University
• Stephen H. Safe, Texas A&M University
• Ana M. Soto, Tufts University
• John J. Stegeman, Woods Hole Oceanographic Institute
• Shanna H. Swan, Univesity of Missouri
• Frederick S. vom Saal, University of Missouri

Committee Charge

• Review critically the literature on HAAs in the environment; identify the known and suspected toxicologic mechanisms and impacts on fish, wildlife, and humans; identify significant uncertainties, limitations of knowledge, and weaknesses in the available evidence; develop a science-based conceptual framework for assessing observed phenomena; and recommend research, monotoring, and testing priorities
• To the extent practicable with available information and study resources, identify particular chemical substances, geographic areas, contaminant sources, human subpopulations, and fish and wildlife populations of special concern with respect to HAAs
• If possible and warranted, suggest general approaches for identifying and mitigating toxicologic problems

Difficulties Encountered

• Limitations and uncertainties in database led different judgments among committee members with regard to:
  • interpreting the general hypothesis
  • determining appropriate sources of information
  • evaluating the evidence
  • defining the agents of concern
  • dose-response relationships
  • periods of susceptibility
  • extrapolation between species
  • evaluating environmental and biologic variables

Committee's Evaluation: Developmental Effects & Reproduction

• Wildlife
  • Examples of effects observed:
    • intersexes in fish exposed to sewage treatment plant effluents
    • testicular feminization in birds exposed to organochlorines
    • gonad and genital abnormalities in alligators of Lake Apopka
  • Unclear if these effects are caused by the hormonal activity of the pollutants

Committee's Evaluation: Developmental Effects & Reproduction

• Humans
  • Reported increases in the incidence of male reproductive disorders (e.g., hypospadias, cryptorchidism, and testicular cancer) cannot be linked to environmental HAAs at this time.
  • Whether sperm concentrations are declining remains controversial.

Committee's Evaluation: Neurologic Effects

• Humans
  • Epidemiology studies have shown associations between prenatal exposure to PCBs and delayed cognitive and behavioral development. Examples of where these observations were made:
    • Populations in Yusho, Japan and Yu-Chang, Taiwan, who were accidentally exposed to high concentrations of PCBs and PCDFs
    • Populations in Michigan and North Carolina, who are exposed via consumption of contaminated fish and other food products

Committee's Evaluation: Immunologic Effects

• Humans
  • Data on the immunologic effects of HAAs are inadequate to support any definitive conclusions.
• Wildlife
  • Evidence of immune suppression from exposure to organochlorines (predominantly PCBs) has been observed in
    • birds of the Great Lakes
    • seals fed fish from the Baltic Sea

Committee's Evaluation: Carcinogenic Effects

• Perinatal exposure to environmental HAAs has not been assessed with respect to carcinogenesis in laboratory animals or humans.
• Humans
  • Available studies conducted to date do not support an association between adult exposure to HAAs and hormonally sensitive cancers.

Committee's Recommendations: Health Effects

• Humans and wildlife exposed to HAAs should continue to be monitored for adverse health effects. Factors that should be considered:
  • life phases of exposed individuals
  • prenatal exposure
  • delayed effects

Committee's Recommendations: Health Effects (continued)

• Laboratory studies should be conducted to determine the short-term and long-term effects of prenatal exposure to HAAs. Cancer studies should also consider latency periods, timing of exposure, and indicators of susceptibility.
• Studies should be conducted to determine whether adverse effects associated with HAAs can be attributed to their hormonal activity.

Committee's Evaluation: Ecological Effects

• The available data indicate that environmental contamination with known HAAs has affected wildlife populations and, in some cases, communities. For example, declines in populations have been observed in:
  • fish and birds of the Great Lakes
  • alligators of Lake Apopka, Florida
  • mink in the U.S.
  • river otters in Europe
  • marine mammals in European waters

Committee's Evaluation: Ecological Effects

• Recommendation
  • Long-term studies of populations exposed to HAA are needed to assess effects on population size, age, structure, and dynamics.

Committee's Evaluation: Exposure

• Better monitoring of contaminated media is needed
• Background concentrations of HAAs in humans need to be established

Committee's Evaluation: Dosimetry

• There is uncertainty whether biologic responses to some HAAs might be greater at low doses than at high doses.
• There are important differences among species and between adult and developing organisms in their responses to HAAs.
• Dose-response relationships need to be investigated at concentrations encountered in the environment.

Committee's Evaluation: Screening and Monitoring

• There are no generally accepted, adequately validated methods for routine identification or monitoring HAAs.
• Most in vitro and in vivo screening assays do not address the full range of putative actions of HAAs.
• The committee agrees, in principle, with the recommendations of EPA's Endocrine Disruptor Screening and Testing Advisory Committee.

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In toxicology, the sequence from exposure to death is: exposure - distribution - target organ - biochemical lesion - microscopic lesion - death of cells - macroscopic evidence of cellular damage - death of the organism.

Classically, the forensic toxicologist has measured the concentration of a given substance in a biological sample, e.g. ethanol in blood. From this scientific measurement, laws have been written concerning driving while intoxicated (DWI). The numbers regarding acceptable levels have change with time. If a certain number of accidents occur when the blood alcohol (BAC) is set at 0.15% (30 years ago), then the law changes to reflect the unacceptable number of accidents associated with a given BAC. The number is a statistical solution to a physiological problem. If we now accept 0.10% in NY and 0.08% in California, we can anticipate a certain level of non-compliance, but we can also see a predictable decrease in the number of accidents. But does the fixed legal limit address individual differences? You know friends who appear to be unaffected by four drinks, the minimum number needed to achieve 0.10% BAC, while others will be obviously out of control at the same BAC. The answer? Biochemical markers such as GABA.

A second example is that smoking's association with lung cancer is a reasonably well established causal relationship. Yet some individuals, exposed only to passive smoke, develop cancer, while some heavy smokers remain reasonably healthy. We need to explore biochemical markers to provide early warning of adverse alterations in cellular activity.

We have journals such as Biological Monitoring and a Committee on Biological markers within the NAS/NRC Board of Environmental Studies and Toxicology. The clinical chemist can pridefully point to a long history of use of biochemical markers in diagnosis, e.g., SGOT, SGTP, and alk. phos., as indicating some liver damage or creatinine and BUN as a measure of kidney function impairment.

As a forensic toxicologist, my earliest recall of our profession addressing this issue was a the 1972 TIAFT Meeting in Glasgow. One of the presenters suggested that classical physical measurements of drug concentrations were not enough. The author(s) suggested that a panel of enzyme tests should be performed. Although the "acceptable range" might be enormous, outliers would become significant indications of unusual toxic responses.

Some biochemical markers might be highly specific. E.g., in the course of vinyl chloride exposure leading to an angiosarcoma, a biochemical lesion would cause the release (or activation of) a biochemical material which would serve as an early warning system. As specific as the angiosarcoma, this marker hopefully would allow the identification of "exposure to a critical amount of vinyl chloride delivered to the liver." Other biomarkers may be more general (non-specific). I will be talking about a peptide material (MDF) which provides information about cardiac involvement in the cause of death; the role of GABA and c-GMP in CNS activity; the measurement of seritonin in trazadone interactions; and cholinesterase activity in phosphorous containing pesticides.

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With more than 100,000 deaths each year and more than 1 million hospitalized, prescription drugs are a major hazard of modern technology. Protecting the public from injury requires a safety chain that begins with initial testing and ends with vigilant and informed consumers. Serious flaws can be identified in each link the safety chain.

Initial Testing: Very little information exists about the long term effects of drugs intended for long term use. There is no provision in law or policy to assure that long-term testing is ever undertaken. Nevertheless many important risks and benefits can only be observed over the long term.

Post-market surveillance: With only 52 employees and a narrowly defined mission, the FDA lacks the money, the staff and the legal mandate to monitor adequately the risks of more than 3,000 different drugs. While staff focuses on the new risks of recently approved drugs, most deaths and serious injuries result from drugs whose dangers are already documented.

Doctor Prescribing: Studies show a large array of problems in the doctors office, with mistakes and misjudgments made with disturbing frequency. Because modern information technology is not used, physicians may be last profession to rely on memory and record their decisions in handwriting.

Serious problems also exist in the pharmacies, which do a poor job in preventing drug interactions, and among consumers, who do not receive adequate information about drugs and often do not take drugs as prescribed.

A complete analysis of the system and recommendations for improving it are contained in the author's new book, Prescription for Disaster (Simon & Schuster, 1998).

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Sulfur mustard [di(2-chloroethyl) sulfide] is a percutaneous, alkylating agent that has been used by warring nations for more than 80 years. Acute topical exposure causes the casualties of such conflicts to develop persistant, incapacitating blisters of the skin.

Among the most intriguing questions about sulfur mustard (HD) toxicity is why basal cells are the primary targets of its vesicating lesions. To investigate this problem replicate cultures of human epidermal keratinocytes (HEK) have been grown from normal skin and exposed to 400 µM HD for 5 min. Using FITC-conjugated antibodies, confocal laser microscopy, and image analyses, it was demonstrated that first passage HEK (maintained in a 0.15 mM Ca²⁺ medium) continued to express keratins K5 and K14 as well as alpha₆beta₄ integrin. K5 and K14 are intermediate filaments and antigenic markers of basal cells. Mutagenic studies have linked K5 and K14 with attachment mechanisms effecting EBS (epidermolysis bullosa simplex), a family of blistering skin diseases.

Acute exposure to HD caused a statistically significant (p < .01) 30% decrease in both K5 and K14 fluorescence within 1 hr of exposure. The loss in K14 expression was progressive and decreased to near zero values within 2 hrs of exposure. This effect occurred well before the expected appearance of in vivo blisters, which have a dose-dependent, clinical latent phase of 8-24 hours.

The effect of HD on K5 fluorescence was not progressive. Acute exposure to HD also caused a statistically significant, early postexposure decrease in alpha₆beta₄ expression associated with an elaborate change in the distribution and organization of these molecules. Mutagenic studies have shown that disruption of alpha₆beta₄ integrin is associated with JEB (junctional epidermolysis bullosa) another important family of blistering skin diseases.

Results indicated that disruption of K5, K14 and alpha₆beta₄ integrin were early events in the HD injury pathway; however, there was no concomitant or obvious effect on cell to substrate attachment through T = 4 hours of post exposure studies.

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An important workshop entitled Standards for Nucleic Acid Diagnostic Applications was held at the NIST Green Auditorium March 15 through 18, 1998. For the first time, NIST provided a forum for all of the communities involved in molecular diagnostic testing. This cross disciplinary dialogue focused on the types of standards needed by these laboratories so that the public can be assured that laboratory results are accurate. Also addressed were the needs for proper quality assurance methods and materials so that testing laboratories may be positioned to provide the best possible results.

Nucleic acid-based diagnostics have revolutionized testing and human identification in forensics, medical molecular genetics, infectious diseases and cancer. With nucleic acids as a central theme for a growing number of diagnostic tests, there is a need for consensus standards for the many tests performed by laboratories in the private and public sectors. The workshop focused on three areas: molecular genetics, cancer diagnostics and infectious diseases. Major issues facing the nucleic acid diagnostic community were discussed and a series of recommendations concerning the management of these issues were developed following productive breakout sessions.

Topics discussed during the meeting included: 1) the role of nucleic acid diagnostics in medical management; 2) novel technologies; 3) the nature of nucleic acid standards in various technologies; 4) automation in nucleic acid analysis and 5) current technologies and problems in the accurate measurement of repeated DNA that occurs with a variety of genetic diseases.

The meeting was sponsored by the NIST Chemical Science and Technology Laboratory as well as the Association of Government Toxicologists, the Centers for Disease Control, the Food and Drug Administration, the National Committee for Clinical Laboratory Standards, and the Health Care Financing Administration. Approximately one hundred scientists representing commercial and federal laboratories as well as teaching and research hospitals were in attendance.

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The Presidential Advisory Committee on Gulf War Veterans' Illnesses was established by the office of the President of the United States in May 1995 to evaluate the government's response to Gulf War veterans' illnesses, the nature of Gulf War veterans' illnesses, and the health effects from Gulf War risk factors. Gulf War risk factors evaluated by the Committee included exposure to pesticides, chemical warfare agents, biological warfare agents, vaccines, pyridostigmine bromide, infectious diseases, depleted uranium, oil-well fire smoke, various occupational exposures, e.g. petroleum products and paints, and psychological and physical stress. The Committee performed a comprehensive review of short and long-term health effects of suspected Gulf War risk factors, at high, low, and chronic exposures, and compared results to Gulf War veterans illnesses. The Committee considered all peer reviewed scientific publications, held discussions with scientists in and out of government, and had more than 12 public Committee meetings with testimony from experts, and review of draft conclusions. After a final public meeting in November 1996 the report was submitted to dozens of outside reviewers both within and outside of the government, and the final report submitted to the President in December 1996.

The Committee concluded that current scientific evidence does not support a causal link between the symptoms and illnesses that most Gulf War veterans report today and exposures to any of the environmental risk factor and commonly suspected Gulf War hazards that the Committee assessed. However, stress, which is known to affect the brain, the immune system, the cardiovascular system, and various hormonal responses, is likely to be an important contributing factor to the broad range of physiological and psychological illnesses currently being reported by Gulf War veterans.

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Humans have used plants as medicine for millennia. During this time, the practice of medicine has evolved from the priest/physician/herbalist to today's specialist. Despite such progress in the theory of medicine, the past 30 years have witnessed an increasing dissatisfaction with its practice. Not coincidentally, during this time period, the herbal industry has undergone an explosion in growth from a few small purveyors of herbal tea to a billion dollar botanical industry. Passage of the Dietary Supplement Health and Education Act (DSHEA) of 1994 has virtually deregulated the sale of botanicals, and this growth has generated a umber of challenges for the industry.

Until recently, the establishment of botanical identity was sufficient to assure quality and safety, and this was possible for most botanicals. With DSHEA and the change in their regulatory status, these products have shifted from being single botanical entities to those containing multiple plant species and plant extracts. These finished products cannot be evaluated using botanical methods alone, and determination of quality and safety fall increasingly to the chemist.

Techniques used for these analyses range from simple spot tests to state of the art chromatographic methods. Applications include simple elimination of the possibility that the product has been contaminated with a poisonous plant (TLC) to determining the plant species by LC "fingerprinting".

In conclusion, quality of botanical products is ensured by detecting toxins which should not be present in the finished product, plant identification (by whatever means) and determination that the level of biologically active constituents falls above limits which define a "quality " product but below those considered toxins.

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