Kenneth W. Turteltaub

LLNL/UC AMS facility and research program

Abstract The Lawrence Livermore National Laboratory (LLNL) and the University of California (UC) now have in operation a large AMS spectrometer built as part of a new multiuser laboratory centered on an FN tandem. AMS measurements are expected to use half of the beam time of the accelerator. LLNL use of AMS is in research on consequences of energy usage. Examples include global warming, geophysical site characterization, radiation biology and dosimetry, and study of mutagenic and carcinogenic processes. UC research activities are in clinical applications, archaeology and anthropology, oceanography, and geophysical and geochemical research. Access is also possible for researchers outside the UC system. The technological focus of the laboratory is on achieving high rates of sample throughput, unattended operation, and advances in sample preparation methods. Because of the expected growth in the research programs and the other obligations of the present accelerator, we are designing a follow-on dedicated facility for only AMS and microprobe analysis that will contain at least two accelerators with multiple spectrometers.

Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines formed during the cooking of meat and fish. Both are genotoxic in a number of test systems and are carcinogenic in rats and mice. Human exposure to these compounds via dietary sources has been estimated to be under 1 microg/kg body wt. per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body wt. per day. We are using accelerator mass spectrometry (AMS), a tool for measuring isotopes with attomole sensitivity, to study the dosimetry of protein and DNA adduct formation by low doses of MeIQx and PhIP in rodents and comparing the adduct levels to those formed in humans. The results of these studies show: 1, protein and DNA adduct levels in rodents are dose-dependent; 2, adduct levels in human tissues and blood are generally greater than in rodents administered equivalent doses; and 3, metabolite profiles differ substantially between humans and rodents for both MeIQx and PhIP, with more N-hydroxylation (bioactivation) and less ring oxidation (detoxification) in humans. These data suggest that rodent models do not accurately represent the human response to heterocyclic amine exposure.

Bioanalytical applications of accelerator mass spectrometry for pharmaceutical research.

Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying isotopes. It has had great impact in the geosciences and is now being applied in the biomedical fields. AMS measures radioisotopes such as 14C, 3H, 41Ca, and 36Cl, and others, with attomole sensitivity and high precision. Its use is allowing absorption, distribution, metabolism and elimination studies, as well as detailed pharmacokinetics, to be carried out directly in humans with very low chemical or radiological hazard. It is used in combination with standard separation methodologies, such as chromatography, in identification of metabolites and molecular targets for both toxicants and pharmacologic agents. AMS allows the use of very low specific activity chemicals (< 1 mCi/mmol), creating opportunities to use compounds not available in a high specific activity form, such as those that must be biosynthesized, produced in combinatorial libraries, or made through inefficient synthesis. AMS is allowing studies to be carried out with agents having low bioavailability, low systemic distributions, or high toxicity where administered doses must be kept low (<1 microg/kg). It may have uses in tests for idiosyncratic metabolism, drug interaction, or individual susceptibility, among others. The ability to use very low chemical doses, low radiological doses, small samples and conduct multiple dose studies may help move drug candidates into humans faster and safer than before. The uses of AMS are growing and its potential for drug development is only now beginning to be realized.

MeIQx-DNA adduct formation in rodent and human tissues at low doses

Heterocyclic amines, such as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), are mutagenic/carcinogenic compounds formed during the cooking of protein-rich foods. Human exposure to MeIQx has been estimated to range from ng/person/day to a few microgram/person/day. In contrast, animal studies have been conducted at doses in excess of 10 mg/kg/day. In order to determine the relevance of high-dose animal data for human exposure, the dose-response curves for [14C]-MeIQx have been determined in rodents at low doses under both single-dose and chronic dosing regimens using the high sensitivity of accelerator mass spectrometry (AMS). To make a direct species comparison, rodent and human colonic MeIQx-DNA adduct levels have been compared following oral administration of [14C]-MeIQx. The results of these studies show: (1) total MeIQx levels are highest in the liver > kidney > pancreas > intestine > blood; (2) MeIQx levels in the liver plateau after 7 days of chronic feeding; (3) hepatic MeIQx-DNA adducts begin to plateau after 2-4 weeks and reach steady-state levels between 4 and 12 weeks on chronic exposures; (4) hepatic DNA adducts generally increase as a linear function of administered dose for a single-dose exposure and as a power function for chronic feeding over a dose range spanning 4 orders of magnitude; (5) human colon DNA adduct levels are approximately 10 times greater than in rodents at the same dose and time point following exposure; and (6) > or = 90% of the MeIQx-DNA adduct in both rodent and human colon appears to be the dG-C8-MeIQx adduct. These studies show that MeIQx is readily available to the tissues for both humans and rodents and that adduct levels are generally linear with administered dose except at high chronic doses where adduct levels begin to plateau slightly. This plateau indicates that linear extrapolation from high-dose studies probably underestimates the amount of DNA damage present in the tissues following low dose. Further, if adducts represent the biologically effective dose, these data show that human colon may be as sensitive to the genotoxic effects of MeIQx as rat liver. The significance of these endpoints to tumor response remains to be determined.

Statistical challenges in the analysis of two-dimensional difference gel electrophoresis experiments using DeCyder™

MOTIVATION The DeCyder software (GE Healthcare) is the current state-of-the-art commercial product for the analysis of two-dimensional difference gel electrophoresis (2D DIGE) experiments. Analyses complementing DeCyder are suggested by incorporating recent advances from the microarray data analysis literature. A case study on the effect of smallpox vaccination is used to compare the results obtained from DeCyder with the results obtained by applying moderated t-tests adjusted for multiple comparisons to DeCyder output data that was additionally normalized. RESULTS Application of the more stringent statistical tests applied to the normalized 2D DIGE data decreased the number of potentially differentially expressed proteins from the number obtained from DeCyder and increased the confidence in detecting differential expression in human clinical studies.

Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH(3)CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

Metabolism of food-derived heterocyclic amines in nonhuman primates.

During the cooking of meats, several highly mutagenic heterocyclic amines (HCAs) are produced. Three HCAs, IQ, MeIQx, and PhIP have been under study for carcinogenicity in cynomolgus monkeys, and to date, IQ has been shown to be a potent hepatocarcinogen. Concomitantly, the metabolic processing of these HCAs has been examined. Metabolism studies show that the potent hepatocarcinogenicity of IQ is associated with the in vivo metabolic activation of IQ via N-hydroxylation and the formation of DNA adducts. In monkeys undergoing carcinogen bioassay with IQ, N-hydroxylation was confirmed by the presence of the N-hydroxy-N-glucuronide conjugate of IQ in urine. The N-hydroxylation of IQ appears to be carried out largely by hepatic CYP3A4 and/or CYP2C9/10, and not by CYP1A2, an isoform not expressed in liver of this species. Notably MeIQx is poorly activated in cynomolgus monkeys and lacks the potency of IQ to induce hepatocellular carcinoma after a 5-year dosing period. The poor activation of MeIQx appears to be due to the lack of constitutive expression of CYP1A2 and an inability of other cytochromes P450, such as CYP3A4 and CYP2C9/10, to N-hydroxylate the quinoxalines. MeIQx is detoxified in monkeys largely by conjugation with glucuronide at the N-1 position. Although the carcinogenicity of PhIP is not yet known, the metabolic data suggest that PhIP will be carcinogenic in this species. PhIP is metabolically activated in vivo in monkeys by N-hydroxylation, as discerned by the presence of the N-hydroxy-N-glucuronide conjugate in urine, bile, and plasma. PhIP also produces DNA adducts that are widely distributed in tissues. The results from these studies support the importance of N-hydroxylation in the carcinogenicity of HCAs in nonhuman primates and by analogy, the importance of this metabolic activation step in the possible carcinogenicity of dietary HCAs in humans.

Effects of Chlorophyll and Chlorophyllin on Low-Dose Aflatoxin B1 Pharmacokinetics in Human Volunteers

Chlorophyll (Chla) and chlorophyllin (CHL) were shown previously to reduce carcinogen bioavailability, biomarker damage, and tumorigenicity in trout and rats. These findings were partially extended to humans, where CHL reduced excretion of aflatoxin B1 (AFB1)-DNA repair products in Chinese unavoidably exposed to dietary AFB1. However, neither AFB1 pharmacokinetics nor Chla effects were examined. We conducted an unblinded crossover study to establish AFB1 pharmacokinetic parameters among four human volunteers, and to explore possible effects of CHL or Chla cotreatment in three of those volunteers. For protocol 1, fasted subjects received an Institutional Review Board–approved dose of 14C-AFB1 (30 ng, 5 nCi) by capsule with 100 mL water, followed by normal eating and drinking after 2 hours. Blood and cumulative urine samples were collected over 72 hours, and 14C- AFB1 equivalents were determined by accelerator mass spectrometry. Protocols 2 and 3 were similar except capsules also contained 150 mg of purified Chla or CHL, respectively. Protocols were repeated thrice for each volunteer. The study revealed rapid human AFB1 uptake (plasma ka, 5.05 ± 1.10 h−1; Tmax, 1.0 hour) and urinary elimination (95% complete by 24 hours) kinetics. Chla and CHL treatment each significantly impeded AFB1 absorption and reduced Cmax and AUCs (plasma and urine) in one or more subjects. These initial results provide AFB1 pharmacokinetic parameters previously unavailable for humans, and suggest that Chla or CHL co-consumption may limit the bioavailability of ingested aflatoxin in humans, as they do in animal models.

The urinary metabolite profile of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is predictive of colon dna adducts after a low-dose exposure in humans

Epidemiologic evidence indicates that exposure to heterocyclic amines in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of heterocyclic amines, which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of heterocyclic amine bioactivation in humans, the most mass abundant heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers where administered a dietary relevant dose of [(14)C]PhIP 48 to 72 hours before surgery to remove colon tumors. Urine was collected for 24 hours after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All 10 subjects were phenotyped for cytochrome P4501A2 (CYP1A2), N-acetyltransferase 2, and sulfotransferase 1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N(2)-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N(2)-glucuronide had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N(2)-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.

Comparison of DNA-adduct and tissue-available dose levels of MeIQx in human and rodent colon following administration of a very low dose

[2‐14C]2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) was administered orally (304 ng/kg body‐weight dose based upon an average 70‐kg‐body‐weight subject) to 5 human colon‐cancer patients (58 to 84 years old), as well as to F344 rats and B6C3F1 mice. Colon tissue was collected from the human subjects at surgery and from the rodents 3.5 to 6 hr after administration. Colon DNA‐adduct levels and tissue available doses were measured by accelerator mass spectrometry (AMS). The mean levels of MeIQx in the histologically normal colon tissue were not different among the human (97 ± 26 pg MeIQx/g), rat (133 ± 15 pg/g) or mouse (78 ± 10 pg/g) tissues; and no difference existed between the levels detected in human normal and tumor tissue (101 ± 15 pg/g). Mean DNA‐adduct levels in normal human colon (26 ± 4 adducts/1012 nucleotides) were significantly greater (p < 0.01) than in rats (17.1 ± 1 adduct/1012 nucleotides) or mice (20.6 ± 0.9 adduct/1012 nucleotides). No difference existed in adduct levels between normal and tumor tissue in humans. These results show that MeIQx forms DNA adducts in human colon at low dose, and that the human colon may be more sensitive to the effects of MeIQx than that of mice or rats. Int. J. Cancer 80:539–545, 1999.