Accomplishments

Drs. Smith and Wiencke (Project 1) and Buffler (Project 2) produced new insights into the natural history and environmental etiology of childhood leukemia. They sequenced various specific DNA changes found in childhood leukemia in 56 patients and showed that these rearrangements could be commonly detected in the neonatal blood spots (Guthrie cards) of the cases. These findings show that most childhood leukemias begin in utero and that maternal and peri-natal exposures are likely to be critical. Indeed, the investigators also found that exposure to indoor pesticides during pregnancy and the first year of life raises leukemia risk, but that later exposures do not. This allows epidemiologists to focus on the critical window of environmental exposure, i.e. pregnancy and peri-natal events, and emphasizes the need to differentiate cytogenetic and molecular sub-groups for studies of causality in childhood leukemia. This work formed the basis of SBRP Research Brief 95: Multidisciplinary Studies of the Origins of Childhood Leukemia (Release Date: 11/06/2002) that is expanded upon further in Core A referring to the following sources:

• Ma, X., P.A. Buffler, R.B. Gunier, G. Dahl, M. Smith, K. Reinier, P. Reynolds, 2002. Critical windows of exposure to household pesticides and risk of childhood leukemia. Environmental Health Perspectives. 110(9): 955-960.
• Wiemels, J.L., Z. Xiao, P.A. Buffler, A.T. Maia, B.M. Dicks, M. Smith, L. Zhang, J. Feusner, J.K. Wiencke, K. Pritchard-Jones, H. Kempski, M.F. Greaves, 2002. In utero origin of t(8;21)AML1-ETO translocations in childhood acute myeloid leukemia. Blood. 99(10): 3801-3804.

The full brief can be found at http://www-apps.niehs.nih.gov/sbrp/researchbriefs/view.cfm?Brief_ID=95
 
Drs. Smith and Wiencke (Project 1) made significant progress in developing methods for measuring chromosome rearrangements, using a technology known as real-time PCR and testing the predictive value and prevalence of these chromosome rearrangements in the general population. They also made significant progress in developing PCR methods for examining the methylation status of cancer-related genes. With investigators from Project 2, a paper was published in Cancer Research showing that the FHIT gene is methylated in a distinct subset of pediatric acute leukemia and may play an important role in the etiology of the acute lymphocytic leukemia.
 
The epidemiologic study of childhood leukemia in Northern and Central California undertaken by Patricia Buffler as Project 2 has helped identify the contribution of various environmental and genetic factors to the risk of childhood leukemia. Preliminary analyses of paternal smoking indicate that pre-conception paternal smoking is associated with a significant increased risk of childhood leukemia. Analyses are underway with complete phase I and II data to explore the contribution of both paternal maternal smoking. Other preliminary findings observed with child's diet (protective effect of orange/bananas; no association with breastfeeding), and increased risks reported with environmental factors such as household pesticide use and paternal smoking.
 
Allan Smith and his group continued his studies of arsenic as part of Project 3. No association was found between ecological measures of arsenic exposure and childhood cancer in Nevada. However, the likelihood that the childhood leukemia cluster near Fallon, Nevada was due to chance was extremely unlikely. His group found increases in respiratory symptoms and diminished pulmonary function testing, and a ten-fold increased risk of bronchiectasis in West Bengal subjects who had arsenic-caused skin lesions. Projects in Chile are showing a very high rate of lung cancer and chronic respiratory disease mortality many years after arsenic exposure in utero and in young children. They found statistically significant differences in arsenic disease susceptibility in India associated with dietary intakes of protein, calcium, fiber, and folate. Their studies comparing diet histories to arsenic methylation in exposed subjects in California and Nevada provided evidence that diet can impact methylation, and people with nutritional deficiencies may be more susceptible to arsenic-caused diseases. Their study in Northern Chile provided no evidence for a natural selection process and showed that arsenic-caused skin lesions can occur despite good nutrition. Other findings included the suggestion that inherited genetic traits can have important influences on individual methylation patterns. No relation between p53 and arsenic exposure was found using gene sequencing of the mutational spectrum in exons 5-8 of the p53 tumor suppressor gene, and p53 expression using histochemical staining.
 
In Project 5, Paul Henderson made significant progress in efforts to develop a novel and ultra sensitive method for the measurement of DNA adducts quantitatively in humans and to study the degree of benzene and trichloroethylene (TCE) macromolecular adduct formation and repair in adults and children at concentrations equivalent to human exposure levels. He succeeded in a proof of principle study using non-labeled ddTTP and synthetic DNA. The enzymatic labeling of the lesion-containing nucleotides using a synthetic construct as a label provided the specificity and high reactivity required in the ¹⁴C-postlabeling of DNA adducts that was previously unattainable.
 
Professors Kent Udell and James Hunt (Project 6) showed that liquid solvents trapped in soils are able to move towards ground water supplies via a process called dense vapor migration. They also demonstrated that steam injection effectively removes these trapped solvents from soils at hazardous waste sites. Initial SBRP funding lead to a very successful full-scale demonstration of Steam Enhanced Extraction at Lawrence Livermore National Laboratory and then to a showcase industrial application at the Visalia Pole Yard in Visalia, California one of the first sites placed on the National Priority List. That project is considered a break-through application of remediation technology, thus earning an EPA Remediation Technology Development Award for "for technical excellence in the development of in situ thermal treatment technologies". The application of Steam Enhanced Extraction at that site decreased the financial liability of the site by $85 million for a cost of about $15 million, showing a clear financial incentive for publicly owned corporations to take a more aggressive approach to environmental restoration.
 
Lisa Alvarez-Cohen and Mark Conrad in Project 8 carried out research pursuant to our Program's objectives of (1) expanding our ability to measure and predict exposures to toxic chemicals through the optimized use of biomarkers, site monitoring, characterization, and modeling; and (2) improving the efficacy and safety of hazardous waste remediation technologies, including incineration and bioremediation. Their field research was the first successful attempt to use stable isotopes to demonstrate the complete in situ biodegradation of chlorinated solvents by enhanced bioremediation. Isotope measurements allowed the tracking and control of the potential generation of harmful biological intermediates. This success will result in approximately $15 million cost savings for the cleanup of this field site, and will open the door for the emerging technique of stable isotope analysis to be applied more widely at a variety of groundwater contaminated sites. This research success formed the basis of SBRP Research brief #93 in 2002 - Full details available at http://www-apps.niehs.nih.gov/sbrp/researchbriefs/view.cfm?Brief_ID=93.
 
Contaminants in freshwater and marine sediments are challenges at many U.S. and international sites. Unlike many hazardous waste sites, sediments accumulate contaminants from many sources over long time periods. Historical reconstruction of human and ecosystem exposure to sediment contaminants is important in anticipating risk from prior exposures. The overall goal of Project 6 led by Professor Hunt was to develop and test a methodology for reconstructing historical exposures to trace metals in estuarine systems. Two sites in the San Francisco Bay Area with significant contamination were used. Members of the research team developed a more complete understanding of transport and mixing in the intertidal zone, including the important contribution of subtidal channels. They found that the superposition of a number of specific forcing frequencies - and the specific phasing between them -determines the redistribution of sediment in the intertidal zone. In studies at the Alameda Naval Air Station field site, which was closed in 1997, they established that the lagoon is a depositional environment that has preserved contaminant records without mixing. Using age-dated sediment cores within the lagoon, they developed a method for trace metal detection using x-ray fluorescence on frozen sediment cores. A 100-cm long sediment core can be resolved at the millimeter scale for a suite of metals in a six-hour period. Since the technique is non-destructive and real-time, locations having interesting results can be subjected to more detailed analysis. The radiochemical and trace metal data collected for the Seaplane Lagoon are a unique data set for demonstrating how to map contaminated estuarine sediments in three dimensions. The results are being shared with the US EPA, the US Navy and their contractors to assist in the RI/FS process.
 
Brenda Eskenazi and Andrew Wyrobek in Project 4 continued their research regarding the transmission of chromosomal and genetic defects via sperm and the effects of paternal host factors (age and diet) and chemical exposure (benzene) on the incidence of physiologic and genetic defective sperm. Their data and review of other studies suggest that fathering children at an older age may be associated with a greater risk for abnormal reproductive outcomes such as reduced fertility, spontaneous abortions, birth defects and genetic diseases resulting from the paternal transmission of chromosomal damage. Specifically, they found a significant age-related decrease in sperm numbers, volume and motility; an age-related increase in the frequency of sperm bearing both the X and Y chromosomes (the type of aneuploidy which could cause Klinefelter syndrome in the offspring) but not other aneuploidies (chromosomes 1, X, Y, 21) in a nation-wide study of fathers of children with Klinefelter syndrome; age-related increases in sperm DNA fragmentation and the frequency of single-stranded sperm DNA breaks but not double-stranded breaks; positive effects of antioxidant intake on sperm motility but no effects on DNA fragmentation; an association between high levels of antioxidant intake and reduced single-stranded sperm DNA breaks; an association between high levels of folate intake and reduced frequency of some types of sperm aneuploidy; and they were able to measure the frequency of mutations in sperm that partially account for the paternal age effect in achondroplasia and Apert syndrome. They also did a comparative study of the effects of age on DNA, genic, chromosomal, and semen-quality endpoints that showed differential effects of age and little evidence of correlation among the endpoints. This suggests different cellular targets and mechanisms are involved in these age effects. In collaboration with Dr. Luoping Zhang of Project 1 and the lab core, samples have been collected from men exposed to benzene in the workplace and matched controls and are currently being analyzed.
 
Martyn Smith and colleagues (Project 1) added new data of value to assessment of the risks associated with benzene exposure. Together with Dr. Stephen Rappaport, of the University of North Carolina-Chapel Hill SBRP, and researchers from the National Cancer Institute and the Chinese Center for Disease Control, they conducted a cross-sectional study in a region near Tianjin, China including 250 shoe workers exposed to benzene-containing glues and 140 unexposed age- and sex-matched controls who worked in three clothes-manufacturing factories. They conducted extensive exposure assessments for 16 months, testing air samples in the factories as well as at each worker's home. Using blood and urine samples, the researchers linked individual air-monitoring data to end-points including white blood cell and platelet counts, lymphocyte subsets and progenitor cell colony formation.
 
As expected, workers exposed to benzene at levels of 1 ppm and higher had fewer total white blood cells, granulocytes, lymphocytes, B cells, and platelets than did unexposed workers. The researchers also found that compared to controls, workers exposed to less than 1 ppm benzene had significantly decreased numbers of all types of white blood cells and platelets. On average, these workers had 15% to 18% fewer granulocytes and B cells than unexposed workers, even after controlling for smoking and other potential confounding factors. The researchers then examined the influences of genetic variation in three enzymes responsible for activating and detoxifying benzene. They found that exposed subjects with variation in two enzymes (MPO and NQO1) were especially susceptible to benzene-induced lowering of white blood cell counts.
 
The results of this study highlight the importance of investigations into long-term health effects, including increased occurrence of blood diseases such as leukemia, in workers exposed to low levels of benzene. This study also shows the importance of careful documentation of exposure levels among all study participants. By repeatedly measuring the personal exposures of the workers in this study, the researchers were able to minimize measurement errors that have obscured effects of low exposure to benzene in most previous investigations.
 
This success formed the basis of SBRP Research brief #121 (http://www-apps.niehs.nih.gov/sbrp/researchbriefs/view.cfm?Brief_ID=121) and referred to the following publication:

• Lan, Q, Zhang, L, Li, G, Vermeulen, R, Weinberg, RS., Dosemeci, M, Rappaport, S M., Shen, Mi Alter, B P., Wu, Y, Kopp, W, Waidyanatha, S, Rabkin, C, Guo, W, Chanock, S, Hayes, RB., Linet, MS., Kim, S, Yin, S, Rothman, N, Smith, MT,, 2004. Hematotoxicity in Workers Exposed to Low Levels of Benzene. Science. 306(5702): 1774-1776.

Dr. Catherine Koshland's group (Project 7) developed chemical kinetic mechanisms describing toxic byproduct production in combustion and waste incineration, demonstrating how post-flame reactions lead to the emission of hazardous chlorinated hydrocarbons. They developed real-time, in situ laser diagnostic techniques, specifically excimer laser fragmentation fluorescence spectroscopy, to measure lead in the gas and particulate phases and in soils with high sensitivity and selectivity. They improved the detection limit for soot particles to 60 ppt, showing that the laser technique can be used to monitor typical combustion exhausts in near real time. The diagnostic is suitable for measuring soot from spark ignition and diesel engine exhaust as well as particles in ambient urban air. They developed a downflow diffusion flame system that produces variable size and concentrations of submicron soot particles. An airblast atomizer for droplets was designed that permits independent variation in the size and spacing. These devices can be used for laboratory and health-related research. Laser-particle interactions were studied to understand fundamental interactions of light with nanoparticles, information that is used to improve small particle diagnostics, and to produce new nanoparticles whose size can be varied by altering the laser parameters. They used a pump-probe laser system to investigate the basic laser-particle interactions, and showed that the fragmentation process is significantly longer than the laser pulse, with changes in the particle properties seen for as long as 500 nsec. Information on the kinetics involved was obtained, and showed that reactions during the first 20 nsec (during the laser pulse) are different from those occurring after the laser pulse. They developed a protocol for burning different coals from the U.S. and China, and showed that the polycyclic aromatic hydrocarbons (PAH) alone are not responsible for the observed lung cancer rates, but that a complex chemical mixture is involved.
 
Work done by the UC Berkeley SBRP's Outreach Core D significantly advanced the goals of the national SBRP to be " proactive in translating the scientific accomplishments emanating from the Program to its stakeholders... to the public through community outreach." Our outreach program, the Children's Environmental Health Network has played a key role in the emergence of pediatric environmental health as a credible and recognized field of research. They have created a national "network" of health professionals, researchers, policy makers and advocates addressing children's environmental health. In addition, they have defined a national research agenda on children's environmental health by convening national conferences at which researchers can share their findings, raise new research questions, and together craft a child?focused national research agenda.

The UC Berkeley program expanded its emphasis on human health in 1995 with the next Superfund reauthorization. Two new epidemiological studies were added. The first, led by Dr. Patricia Buffler, addressed childhood leukemia, which has been steadily increasing in the US population since the early 1970s. The case-control study recruited 200 children in Northern California and is now investigating environmental exposures to pesticides and household solvents. The study is also looking for biomarkers of susceptibility and effect. Drs. Smith and Wiencke provide the laboratory component of the study. Recently, Dr. Buffler was awarded an RO1 grant of $4.5 million to expand the study to include more children.
 
The second epidemiological study targeted a new area: reproductive health. This signaled the expansion of UC Berkeley's program from a sole focus on cancer to an examination of other health endpoints that result from environmental exposures. Under the leadership of Drs. Brenda Eskenazi and Andrew Wyrobek, the study examines the role of genetic damage in the fathers gametes in producing health effects in the offspring. It utilizes biomarkers of genetic damage in sperm developed by Dr. Andrew Wyrobek at Lawrence Livermore National Lab. These biomarkers can be used to potentially identify reproductive toxicants. Dr. Eskenazi was recently awarded $6 million from the NIEHS and EPA to establish a Center of Excellence in Children's Environmental Health at UC Berkeley.
 
The 1995 reauthorization also brought the addition of an outreach core with a focus on children's health, This program was called the Children's Environmental Health Network. Headed by Joy E. Carlson, this program promotes issue awareness and trains health professionals to recognize and treat environmental health problems in children. It has had a major impact on promoting this issue at the National level. Another new program was the analytical core, under the guidance of Dr. Robert Spear, to provide statistical and computational guidance to the other researchers in the program. This has helped link the researchers electronically and incorporate the latest statistical methods into their research.

The Superfund Program reauthorization in 1992 led to new directions for the UC Berkeley program. The successful completion of three projects meant the program could expand as the team prepared to add another research tool to their investigations-epidemiology.
 
Major findings:

• Dr. Allan Smith leads a study of lung and bladder cancer patients who were exposed to naturally occurring arsenic in drinking water. His population-based studies in Nevada and Chile uncover a high incidence in bladder cancers from arsenic exposure, and he shows that methylation does not protect from arsenic-related carcinogenesis at low levels of exposure. Using a novel micronucleus assay developed in the laboratory of Dr. Martyn Smith, the researchers are the first to demonstrate that concentrations of arsenic in drinking water at the US standard of 50 ppb produced increased levels of genetic damage in bladder epithelial cells of humans who drink the water. They go on to perform an intervention study that shows a decrease in genetic damage in the bladder following a reduction in arsenic exposure.
• Using accelerator mass spectrometry (AMS), Dr. Ken Turteltaub detects DNA adduct formation in bone marrow and liver following exposure to very low doses of benzene and trichloroethylene.
• Drs. Martyn Smith and Alma Burlingame continue their research on biomarkers of benzene exposure, and add trichloroethylene and arsenic biomarkers to their research agendas.
• Dr. John Wiencke discovers the molecular basis for genetic susceptibility to diepoxybutane, a metabolite of 1,3-butadiene, an important industrial chemical and pollutant used in the manufacture of synthetic rubber.
• Dr. Kent Udell completes a full-scale demonstration of steam injection for contaminant removal.
• Dr. Koshland develops an in situ real time technique for detecting and monitoring chlorinated hydrocarbons in incinerators that has since been extended to ammonia and several metals.

Goals: 1) To develop new mathematical modeling and pharmacokinetic techniques for use in human health risk assessment; and 2) To find biological markers that could be used to determine prior exposures and quantify the doses of these exposures discovered.
 
Major findings:

• Dr. Martyn Smith's lab reports that benzene's phenolic metabolites cause synergistic aneuploidy in human lymphocytes, leading to mechanistic hypothesis for benzene's carcinogenicity.
• Dr. Stephen Rappaport develops a simple and inexpensive method of measuring protein adducts from styrene oxide and other electrophillic metabolites using Raney Nickel.
• Dr. John Wiencke at UCSF finds a strong correlation between genetic enzymatic deficiencies and induction of sister chromatid exchange.
• Dr. William Bodell uses 32P-postlabeling to identify DNA adducts and, with Dr. Alma Burlingame, identifies specific adducts formed from exposure to styrene and benzoquinone, a toxic metabolite of benzene.
• In modeling and pharmacokinetic research, Dr. Robert Spear develops a physiologically-based pharmacokinetic model of benzene metabolism.
• Dr. Thomas Tozer develops a pharmacokinetic model for perchloroethylene and discovers that pentachlorophenol exposure leads to persistent metabolic breakdown products.
• In 1988 four additional research projects established which focus on the physical and chemical transformation of toxins in the environment.
• Drs. James Hunt and Kent Udell show that liquid solvents trapped in soils are able to move towards ground water supplies via a process called dense vapor migration. They also demonstrate that steam injection effectively removes these trapped solvents from soils at hazardous waste sites.
• Drs. John Harte and Susan Anderson develop a battery of genotoxicity tests using three aquatic species.
• Dr. Joan Daisey suceeds in modeling the migration of pollutants from soil gas into homes.
• Dr. Catherine Koshland identifies key conditions for toxic byproduct production in combustion and waste incineration, showing that at sub-optimal temperatures relatively innocuous compounds such as ethyl chloride can be converted into highly hazardous chemicals such as vinyl chloride.