Attomole Detection of 3H in Biological Samples Using Accelerator

Biology and Biotechnology Research Program and Center for Accelerator Mass Spectrometry .... Environmental Science & Technology 2002 36 (13), 2848-285...
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Chem. Res. Toxicol. 1998, 11, 1217-1222

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Attomole Detection of 3H in Biological Samples Using Accelerator Mass Spectrometry: Application in Low-Dose, Dual-Isotope Tracer Studies in Conjunction with 14C Accelerator Mass Spectrometry Karen H. Dingley,* Mark L. Roberts, Carol A. Velsko, and Kenneth W. Turteltaub Biology and Biotechnology Research Program and Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California 94551-9900 Received June 19, 1998

This is the first demonstration of the use of accelerator mass spectrometry (AMS) as a tool for the measurement of 3H with attomole (10-18 mol) sensitivity in a biological study. AMS is an analytical technique for quantifying rare isotopes with high sensitivity and precision and has been most commonly used to measure 14C in both the geosciences and more recently in biomedical research. AMS measurement of serially diluted samples containing a 3H-labeled tracer showed a strong correlation with liquid scintillation counting. The mean coefficient of variation of 3H AMS based upon the analysis of separately prepared aliquots of these samples was 12%. The sensitivity for 3H detection in tissue, protein, and DNA was approximately 2-4 amol/mg of sample. This high sensitivity is comparable to detection limits for 14C-labeled carcinogens using 14C AMS and demonstrates the feasibility of 3H AMS for biomedical studies. One application of this technique is in low-dose, dual-isotope studies in conjunction with 14C AMS. We measured the levels of 3H-labeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 14C-labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rat liver tissue and bound to liver DNA and protein 4.5 h following acute administration of individual or coadministered doses in the range of 4-5100 pmol/kg of body weight. Levels of PhIP and MeIQx in whole tissue and bound to liver protein were dose-dependent. MeIQx-protein and -DNA adduct levels were higher than PhIP adduct levels, which is consistent with their respective carcinogenicity in this organ. Coadministration of PhIP and MeIQx did not demonstrate any measurable synergistic effects compared to administration of these compounds individually. These studies demonstrate the application of AMS for the low-level detection of 3H in small biological samples and for its use in conjunction with 14C AMS for dual-labeling studies.

Introduction 3

H is a widely used radioisotope in biological tracing studies, largely due to the low cost and ease of synthesis of 3H-labeled compounds. Although the most common method for 3H quantification in biological samples is liquid scintillation counting, this technique requires large samples and counting times of several hours for the most sensitive measurements. Additional problems include a low counting efficiency and, depending upon the sample composition and size, chemiluminescence and quenching (1). Provisional studies using a two-step system for sample preparation have demonstrated that accelerator mass spectrometry (AMS)1 could, in principle, be used to detect 3H in biological samples with a 100-1000-fold improvement over decay counting techniques (2). Consequently, the aim of this study was to demonstrate the use of AMS as a tool for measuring 3H-labeled carcino* To whom correspondence and requests for reprints should be addressed. 1 Abbreviations: AMS, accelerator mass spectrometry; CI, confidence interval; CV, coefficient of variation; HCA, heterocyclic amine; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; PhIP, 2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine.

gens in whole tissue and covalently bound to protein and DNA in microgram to milligram quantities of material following low-dose exposures. AMS was initially developed for use in earth and environmental sciences to quantify rare, long-lived isotopes in small samples (reviewed in ref 3). Recently, it has been used to measure 14C-labeled compounds with high sensitivity and precision in biomedical studies (4). This has facilitated investigations of the dosimetry of environmentally relevant exposure levels of 14C-labeled drugs and carcinogens in laboratory animals and humans (5-7). The development of procedures for measuring 3H in small biological samples by AMS enables low-level tracer studies to be conducted with chemicals not readily available in a 14C-labeled form. In addition, due to the versatility of AMS, it is possible to measure both 14C- and 3 H-labeled compounds. Consequently, the metabolism and distribution of two independent tracers can be investigated at low doses, a situation more relevant to human exposures, which are typically from multiple compounds (8). The heterocyclic amines (HCAs) 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-

10.1021/tx9801458 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/26/1998

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Dingley et al. Table 1. Doses of [3H]PhIP and [14C]MeIQx Administered to Male F344 Rats by Gavage

Figure 1. Structures of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (a) and 2-amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx) (b) and the positions of isotope labels (*).

dimethylimidazo[4,5-f]quinoxaline (MeIQx) (Figure 1) are formed during the cooking of meat (9) and are multiorgan carcinogens in rodents. In rats, MeIQx administration causes mainly liver tumors (10), whereas PhIP induces colon, prostate, and mammary tumors (11, 12). Humans are exposed to many HCAs simultaneously in the diet; hence, it is important to study the effects following coexposure to these compounds, which could potentially alter the cancer risk. In fact, chronic exposure of rats to five HCAs in the diet resulted in significantly higher levels of tumor induction than was predicted from singlecompound studies (13). This synergistic effect is supported by evidence from medium-term liver bioassay studies in rats, which demonstrated enhanced liver cell foci formation following chronic exposure to several heterocyclic amines compared to that for exposure to individual compounds (14, 15). The mechanism for this apparent synergy is poorly understood, although it is likely to result from enzyme induction or alterations in the amount of the compound(s) reaching the target site. Therefore, in this study, we have used 3H AMS, in conjunction with 14C AMS, in an initial proof of principle experiment to investigate whether coadministration of these compounds may alter tissue-available doses or binding to macromolecules such as DNA and protein following acute low-dose exposures.

Experimental Procedures Caution: PhIP and MeIQx are carcinogenic to rodents and should be handled with care. Materials. [ring-G-3H]PhIP (see Figure 1 for the positions of the 3H labels) was purchased from ChemSyn Science Laboratories (Lenexa, KS). [3H]PhIP was purified isocratically using HPLC by elution from a C18 reversed phase column with 50% water/50% methanol at a flow rate of 0.8 mL/min with the UV absorbance monitored at 315 nm. The purified [3H]PhIP had a specific activity of 9.05 Ci/mmol. An aliquot of purified [3H]PhIP was reanalyzed by HPLC in conjunction with scintillation counting of the HPLC fractions to determine radiopurity, which was 90%, implying that some of the 3H label was labile. [2-14C]MeIQx (Figure 1) was obtained from Toronto Research Chemicals (Toronto, ON). Similarly, [14C]MeIQx was purified using HPLC by elution from a C18 reversed phase column with

animal number

[3H]PhIP dose (pmol/kg of bw)

[14C]MeIQx dose (pmol/kg of bw)

1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24 25-27 28-30 31-33 34-36 37-39

0 0 0 0 0 4.0 44 440 4500 4.2 43 430 5100

0 4.4 44 440 4600 0 0 0 0 4.5 43 480 4100

a 0 to 100% methanol/water gradient at a flow rate of 1 mL/ min over the course of 30 min with the UV absorbance monitored at 273 nm. The purified [14C]MeIQx had a specific activity of 45.57 mCi/mmol and a radiopurity of 97%. All other chemical reagents were of analytical grade. Laboratory Animal Dosing and Sample Collection. Male F344 rats (200-250 g) were obtained from Simonsen Laboratories (Gilroy, CA). Animals were housed three per cage, kept on a 12 h light/dark cycle, maintained at 24 °C, and given water and food ad libitum. The food, but not water, was removed 12 h prior to dosing. [14C]MeIQx and [3H]PhIP doses were administered in acidified water, with animals receiving 2 µL of dosing solution/g of body weight (bw) by gavage. The exact concentration of radiolabeled HCA in each of the dosing solutions was determined by liquid scintillation counting of 10100 µL aliquots. In all cases, the doses coadministered differed from the doses administered alone by less than 14%. Three groups of 12 rats were used (four doses per group and three animals per dose), and three controls which received acidified water alone. The first group was dosed with [14C]MeIQx in the dose range of 4.4-4600 pmol/kg of bw. The second group was administered [3H]PhIP in the dose range of 4-4500 pmol/kg of bw. The third group received both 4.5-4100 pmol/kg of bw [14C]MeIQx and 4.2-5100 pmol/kg of bw [3H]PhIP (Table 1). Four and one-half hours after dosing, rats were euthanized by carbon dioxide asphyxiation and the livers removed and stored at -35 °C. Extraction of DNA and Protein from Liver Tissue. DNA was extracted from liver tissue using Qiagen DNA extraction columns (Qiagen, Chatsworth, CA), as previously described (17). DNA was quantified by measuring the absorbance at 260 nm, assuming an absorbance value of 1 was equivalent to a DNA concentration of 50 µg/mL. DNA purity was determined from the 260 nm/280 nm absorbance ratio. DNA with a ratio of 1.71.8 was used for AMS analysis. Protein was extracted as follows. One gram aliquots of liver tissue were homogenized and then lysed in 5 mL of lysis buffer [4 M urea containing 1% (v/v) Triton X-100, 10 mM EDTA, 100 mM NaCl, 10 mM TrisHCl (pH 8), and 10 mM dithiothreitol] overnight at 37 °C in a shaking water bath. Lysates were centrifuged at 3000g for 15 min to remove incompletely lysed tissue. Perchloric acid [500 µL, 70% (v/v)] was added to the supernatant to precipitate proteins. Samples were centrifuged at 3000g for 15 min, and the resultant pellets containing protein were washed sequentially with 5% (v/v) perchloric acid, twice with 50% (v/v) methanol, and twice with 1/1 ether/ethanol. Washed pellets were air-dried overnight. This procedure does not produce a pure protein extract, as samples will contain other acidprecipitable macromolecules. Aliquots of protein and DNA were analyzed by AMS, as described below. Sample Preparation and 3H AMS Measurement. A twostep technique was used to convert the hydrogen in the organic samples into titanium hydride, a solid form which is compatible with the AMS ion source. Tissue, DNA, or protein (0.3-6 mg) was placed into a quartz tube and completely dried in a

Tritium Analysis by Accelerator Mass Spectrometry centrifugal evaporator. The dry sample was sealed under vacuum and then oxidized to water and carbon dioxide using copper oxide in a sealed combustion tube (16). Water was cryogenically separated and transferred under vacuum to a Pyrex tube containing 200 mg of zinc and 10 mg of titanium powder, reduced to hydrogen gas, and reacted with titanium, making titanium hydride, which was then loaded into aluminum AMS sample holders, and 3H/1H ratios were measured by AMS (2; Carol A. Velsko and Mark L. Roberts, manuscript in preparation). The 3H/1H ratios were used to calculate either femtomoles of PhIP per gram of tissue or protein or DNA adduct levels based upon the specific activity of the [3H]PhIP, the percentage (w/w) of hydrogen (1H) in the sample, and the percentage weight loss upon drying (tissue only), following subtraction of the 3H/1H ratio of the control samples. The average percentage (w/w) of hydrogen in dry liver tissue (7.5%), protein (4.9%), and DNA (3.9%) and the average weight loss of liver tissue following drying (66%) were determined by analyzing aliquots of sample using a C:N:S analyzer (Carlo-Erba Nitrogen NA 1500 Series 2 analyzer, Milan, Italy). Serially diluted tributyrin samples containing [3H]PhIP were used to assess the accuracy, precision, and limit of detection of the sample preparation and AMS measurement process. Tributyrin (C15H26O6, 1.032 g/mL) is an organic, nonvolatile liquid. The 3H/1H ratios were determined by liquid scintillation counting of 52-516 mg aliquots in triplicate for 10 min each (1 mol of 3H has 6.41 × 1016 dpm) using a Pharmacia Wallac 1410 liquid scintillation counter (Gaithersburg, MD). For 3H AMS, at least three aliquots of 2 mg of each sample were combusted and transferred and the 3H/1H ratios measured. Sample Preparation and 14C AMS Measurement. Biological samples for 14C AMS measurement were converted to graphite and the 14C/13C ratios determined by AMS, as previously described (4, 16, 17). Prior to analysis, 2 mg of tributyrin was added to each DNA sample (0.05-0.3 mg) to provide the carbon content necessary for efficient graphitization. Aliquots of 5-10 mg of tissue or protein were graphitized without addition of tributyrin. The 14C/13C ratios were then used to calculate either femtomoles of MeIQx per gram of tissue or protein or DNA adduct levels based upon the specific activity of the [14C]MeIQx and the percentage (w/w) of carbon in the sample following the subtraction of the 14C contribution from control samples and any added tributyrin. The average percentage (w/w) of carbon in wet liver tissue (17%), protein (28.3%), and DNA (29%) was determined using a C:N:S analyzer. Statistics. The 3H/1H ratios obtained for the tributyrin samples by liquid scintillation counting represent the mean ( SD of three samples. The 3H/1H ratios obtained for the tributyrin samples by 3H AMS represent the weighted mean ( the greater of the SD or the AMS measurement error of at least three samples. The AMS measurement error represents the spread of repeated isotope ratio determinations. Errors associated with the weighted means were calculated using the Pythagorean formula (i.e., the square root of the sum of the squares of the errors associated with the individual measurements). The animal data represent one AMS measurement per rat sample ( the measurement error. Dose-response data were log-transformed and then analyzed by ordinary linear regression. Estimated dose-response coefficients were compared using appropriate terms in a multiple linear regression on the log-transformed data. Coefficients were considered statistically different with a P value of 0.05). Similarly, multiple regression analysis of the dose-response data showed that MeIQx-protein adduct levels were 5.8-fold higher than PhIP-protein adduct levels (95% CI of 2-16.2). Coadministration resulted in no significant difference in PhIP-protein or MeIQx-protein adduct levels compared to individual dosing (P > 0.05).

In both dosing regimens, binding of [3H]PhIP or [14C]MeIQx to DNA was only detectable above controls at the highest dose (Table 3). The extent of binding of MeIQx to liver DNA at this dose was significantly greater than the extent of PhIP binding to DNA (P < 0.001), with MeIQx-DNA adduct levels approximately 30-fold higher than PhIP-DNA adduct levels. There was no significant difference between MeIQx-DNA adduct levels detected upon coadministration, compared to administration of MeIQx alone (P > 0.05). PhIP-DNA adduct levels were lower following coadministration (P ) 0.02).

Discussion This study describes the first use of 3H AMS in a biological tracer experiment. Using this technique, it has been possible to quantify low attomole (10-18 mol) amounts of a 3H-labeled tracer in microgram to milligram sample sizes. When used in conjunction with 14C AMS, it allows the measurement of 3H and 14C in parallel in biological samples from the same source with high sensitivity. 3H AMS measures the 3H/1H ratio of a sample, which is then used to calculate the 3H content. By measuring ratios rather than radioactive decay, AMS can increase the detection limits of long-lived isotopes such as 3H (halflife of 12.3 years) (3). On the basis of a signal/noise ratio of 2, the detection limit of 3H AMS for the measurement of 3H in tissue, protein, and DNA was approximately 2-4 amol/mg of sample. In comparison, this level of sensitivity would not be possible with traditional liquid scintillation counting unless a large amount of material was available. Although this study mainly used sample sizes of 2-6 mg, it was possible to obtain a significant 3H and 1H signal from 0.3 mg of DNA. The lowest DNA adduct level detected was approximately two adducts per 1012 normal nucleotides, which produced a 3H/1H ratio 7 times

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Table 3. Liver DNA Adduct Levels Following Acute Oral Administration of [3H]PhIP and [14C]MeIQx Alone or Coadministered

a

animal number

[3H]PhIP dose (pmol/kg of bw)

[14C]MeIQx dose (pmol/kg of bw)

PhIP-DNA adduct level (adducts/1012 nucleotides)a

MeIQx-DNA adduct level (adducts/1012 nucleotides)a

13 14 15 25 26 27 37 38 39

0 0 0 4500 4500 4500 5100 5100 5100

4600 4600 4600 0 0 0 4100 4100 4100

9.1 ( 0.7 12.8 ( 0.8 13.8 ( 0.7 6.9 ( 0.8 3.6 ( 0.7 2.2 ( 0.4

278.4 ( 5.3 363.0 ( 6.0 222.2 ( 5.0 215.1 ( 5.2 304.9 ( 6.5 138.6 ( 4.5

The data represent one measurement per sample ( measurement error.

higher than that for control DNA. This sensitivity is several orders of magnitude higher than that of alternative analytical techniques for measuring DNA adducts, such as 32P-postlabeling (18), and is comparable to the detection limit for measuring 14C-labeled carcinogens using 14C AMS (4). On the basis of the preparation and analysis of replicate tributyrin samples by 3H AMS, the mean CV was 12%. In contrast, the precision for measurement of multiple aliquots of a sample containing 14C by AMS is routinely 5-10% (4) and implies that further improvements in 3H AMS sample preparation could be made. However, the instrument precision in measuring the 3H samples was high (mean CV of 4-6%), with improvements expected using a dedicated 3H AMS machine. Currently, our AMS machine is used to study a number of isotopes and is not fully optimized for measuring 3H. The major concern with using 3H-labeled compounds in tracer experiments is 3H exchange. This may result in loss of isotope label and underestimation of the amount of compound in the samples. In this study, biological samples were not analyzed for evidence of 3H exchange, although in an attempt to minimize this potentially major problem, the [3H]PhIP used here was HPLC purified immediately prior to use. In the future, due to the higher sensitivity of 3H AMS compared to that of decay counting, tracers with labels synthesized in more specific, stable positions of the molecule could potentially be employed. Although the biological applications of 3H and 14C AMS in dual-isotope labeling studies are likely to be widespread, ongoing research in our laboratory is currently aimed at identifying the carcinogenic and toxicological effects of the HCAs. The consequences of human exposure to these compounds in the environment are yet to be established, although previous experiments using 14C AMS have determined bioavailability and DNA adduct levels in tissues following environmentally relevant doses of individual HCAs in both rodents and humans (6, 7, 19, 20). However, dietary exposure is likely to involve complex mixtures of compounds; hence, it is important to understand the dose-response relationships following multicompound exposures. This issue is supported by studies that showed a synergistic effect of HCAs in the induction of tumors and preneoplastic foci in the liver (13-15). The molecular basis of this apparent synergy remains uncertain, although it is possible that HCAs interact to modify one another’s absorption, metabolism, or excretion. Furthermore, compounds may compete for enzyme active sites or cause enzyme induction. It is likely that such interactions are dependent upon the dosing regimen and exposure dose (8, 21). This is

supported by the observation that induction of liver foci by mixtures of HCAs demonstrated a dose-dependent relationship, with no effect observed at the lowest dose examined. Furthermore, the effect was more pronounced with mixtures of ten HCAs, rather than five (15). However, the possibility of an interaction between these compounds at low doses leading to a modulation in bioavailability and adduct levels has yet to be investigated. As a proof of principle for 3H AMS, bioavailability and protein and DNA adduct levels in the liver of rats dosed acutely with [3H]PhIP and/or [14C]MeIQx were determined at doses relevant to human exposure. Measurement of rat liver tissue samples following exposure to low doses of [3H]PhIP or [14C]MeIQx alone revealed dose-dependent levels of HCA, with levels consistent with previous studies conducted with 14Clabeled compounds (17, 20). Four and one-half hours following acute exposure, the levels of MeIQx in the liver tissue were approximately 2-fold higher than those of PhIP, suggesting a greater bioavailability of MeIQx at this time point. No measurable alterations in the levels of these HCAs were observed upon coadministration. Consequently, it appears that PhIP and MeIQx do not interact directly, leading to altered absorption, distribution to the liver, or clearance from the liver. The HCAs, including PhIP and MeIQx, are metabolically activated principally by cytochrome P450 1A2, by conversion of the exocyclic amino group to a hydroxyamino group. This group can be further enzymatically activated by conjugation, for example to acetate or sulfate, forming what are considered to be the ultimate genotoxic metabolites, which may bind to protein and DNA-forming covalent adducts (22-24). At the dose levels for which covalent binding to liver protein was detectable, the extent of protein adduct formation was dose-dependent. This is consistent with previous DNA adduct dose-response studies conducted with these compounds at low doses (4, 17, 20). Significantly, although PhIP and MeIQx can be metabolically activated to genotoxic metabolites by common enzymatic pathways, they show differing target organ specificity in rats, with MeIQx causing liver tumors but PhIP causing colon, breast, and prostate tumors (10-12). This distinction is consistent with the relative liver protein adduct levels detected for these compounds, which were approximately 6-fold greater for MeIQx than for PhIP. Furthermore, the extent of binding of MeIQx to liver DNA at the highest dose was approximately 30-fold greater than that of PhIP. Therefore, the relative levels of protein and DNA adducts found in the liver shortly after exposure are consistent with the relative hepatocarcinogenicity of

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these compounds. Similar studies are planned to investigate if adduct levels in the colon, breast, and prostate are related to the organotropy of these HCAs. As PhIP and MeIQx share common metabolic activation pathways, it is possible that coadministration results in altered metabolism compared to that with individual dosing. This possibility is considered remote at low doses because enzyme pathways are unlikely to be saturated and enzyme induction is unlikely to occur in the time frame of this acute dosing study. Consistent with this theory, no difference was observed in protein adduct levels between animals exposed to an individual dose of PhIP or MeIQx compared to those dosed by coadministration. PhIP-DNA adduct levels appeared to decrease with coadministration, which may suggest a reduction in the levels of genotoxic PhIP metabolites, or that the capacity to repair PhIP-DNA adducts was increased by MeIQx coadministration. However, due to the small number of samples with detectable DNA adduct levels and the fact that this observation is not consistent with protein adduct levels, this result should be viewed with caution and warrants further investigation. In this acute study, no synergy was observed between PhIP and MeIQx at low doses. This contrasts with the synergy observed in chronic, high-dose carcinogenicity and liver bioassay studies (13-15), but is consistent with the observation that such effects are dose-dependent and related to the number of compounds employed (15). Therefore, it is likely that the synergistic effects are due to saturation of enzyme pathways or the induction of enzymes involved in metabolic activation. For example, chronic exposure to heterocyclic amines at high doses over 1-7 days has been shown to result in P450 1A induction (reviewed in ref 25). Furthermore, our study was conducted with only two compounds, not with groups of five or ten HCAs. Therefore, to further investigate the cause of this synergy, similar dual-isotope tracer experiments need to be conducted with other HCA combinations over wider dose ranges in acute as well as chronic studies.

Acknowledgment. This paper is dedicated to the memory of Caroline Holloway. We thank Kurt Haack for AMS sample preparation and David Nelson for advice with statistics. The studies described were performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory (Contract W-7405-ENG-48) and partially supported by NIH (Grants CA55861 and ESO 04705) and USAMRMC (Grant MM4559FLB).

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