Cliff Pereira

Polycyclic aromatic hydrocarbons as skin carcinogens: comparison of benzo[a]pyrene, dibenzo[def,p]chrysene and three environmental mixtures in the FVB/N mouse.

The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by ³²P post-labeling, did not correlate with tumor incidence. PAH-dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p<0.05), showed DBC and BaP to cluster distinct from PAH mixtures and each other. BaP and mixtures up-regulated phase 1 and phase 2 metabolizing enzymes while DBC did not. The carcinogenicity with DBC and two of the mixtures was much greater than would be predicted based on published Relative Potency Factors (RPFs).

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.

Genomic Profiling Reveals an Alternate Mechanism for Hepatic Tumor Promotion by Perfluorooctanoic Acid in Rainbow Trout

Background Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are considered refractory to carcinogenesis by PPs. Previous studies with rainbow trout indicate they are also insensitive to peroxisome proliferation by the PP dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. Objectives In this study, we used trout as a unique in vivo tumor model to study the potential for PFOA carcinogenesis in the absence of peroxisome proliferation compared with the structurally diverse PPs clofibrate (CLOF) and DHEA. Mechanisms of carcinogenesis were identified from hepatic gene expression profiles phenotypically anchored to tumor outcome. Methods We fed aflatoxin B1 or sham-initiated animals 200–1,800 ppm PFOA in the diet for 30 weeks for tumor analysis. We subsequently examined gene expression by cDNA array in animals fed PFOA, DHEA, CLOF, or 5 ppm 17β-estradiol (E2, a known tumor promoter) in the diet for 14 days. Results PFOA (1,800 ppm or 50 mg/kg/day) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity (p < 0.0001), whereas CLOF showed no effect. Carcinogenesis was independent of peroxisome proliferation, measured by lack of peroxisomal β-oxidation and catalase activity. Alternately, both tumor promoters, PFOA and DHEA, resulted in estrogenic gene signatures with strong correlation to E2 by Pearson correlation (R = 0.81 and 0.78, respectively), whereas CLOF regulated no genes in common with E2. Conclusions These data suggest that the tumor-promoting activities of PFOA in trout are due to novel mechanisms involving estrogenic signaling and are independent of peroxisome proliferation.

Protection by dietary zinc in ALS mutant G93A SOD transgenic mice

Mutations to the copper, zinc superoxide dismutase (SOD) gene are responsible for 2-3% of amyotrophic lateral sclerosis (ALS) cases. These mutations result in the protein having a reduced affinity for zinc. SOD becomes toxic to motor neurons when zinc is missing from its active site. Recently, high dosages of zinc (75 and 375 mg/kg/day) have been paradoxically reported to increase the death of G93A-mutant SOD transgenic mice [G.J. Groeneveld, J. de Leeuw van Weenen, F.L. van Muiswinkel, H. Veldman, J.H. Veldink, J.H. Wokke, P.R. Bar, L.H. van den Berg, Zinc amplifies mSOD1-mediated toxicity in a transgenic mouse model of amyotrophic lateral sclerosis, Neurosci. Lett. 352 (2003) 175-178]. In contrast, we have found that moderate supplementation of zinc (approximately 12 mg/kg/day) delayed death in G93A-mutant SOD mice by 11 days compared to mice on a zinc-deficient diet. Supplementing zinc with even 18 mg/kg/day resulted in a more rapid death of some mice, consistent with the results of Groenevelt et al. However, large amounts of zinc competitively inhibit copper absorption, which inhibits the copper-dependent ceruloplasmin, and can cause a lethal anemia. We found that supplementing the 18 mg/kg/day dosage of zinc with 0.3 mg/kg/day of copper prevented the early death from zinc treatment alone. These data support a role for moderate levels of dietary zinc potentially protecting against the toxicity of ALS-associated SOD and the protection does not result from depleting copper.

Comparative Metal Oxide Nanoparticle Toxicity Using Embryonic Zebrafish

Engineered metal oxide nanoparticles (MO NPs) are finding increasing utility in the medical field as anticancer agents. Before validation of in vivo anticancer efficacy can occur, a better understanding of whole-animal toxicity is required. We compared the toxicity of seven widely used semiconductor MO NPs made from zinc oxide (ZnO), titanium dioxide, cerium dioxide and tin dioxide prepared in pure water and in synthetic seawater using a five-day embryonic zebrafish assay. We hypothesized that the toxicity of these engineered MO NPs would depend on physicochemical properties. Significant agglomeration of MO NPs in aqueous solutions is common making it challenging to associate NP characteristics such as size and charge with toxicity. However, data from our agglomerated MO NPs suggests that the elemental composition and dissolution potential are major drivers of toxicity. Only ZnO caused significant adverse effects of all MO particles tested, and only when prepared in pure water (point estimate median lethal concentration = 3.5–9.1 mg/L). This toxicity was life stage dependent. The 24 h toxicity increased greatly (∼22.7 fold) when zebrafish exposures started at the larval life stage compared to the 24 h toxicity following embryonic exposure. Investigation into whether dissolution could account for ZnO toxicity revealed high levels of zinc ion (40–89% of total sample) were generated. Exposure to zinc ion equivalents revealed dissolved Zn2+ may be a major contributor to ZnO toxicity.

The influence of diesel exhaust on polycyclic aromatic hydrocarbon-induced DNA damage, gene expression and tumor initiation in Sencar mice in vivo

The carcinogenic effects of individual polycyclic aromatic hydrocarbons (PAH) are well established. However, their potency within an environmental complex mixture is uncertain. We evaluated the influence of diesel exhaust particulate matter on PAH-induced cytochrome P450 (CYP) activity, PAH-DNA adduct formation, expression of certain candidate genes and the frequency of tumor initiation in the two-stage Sencar mouse model. To this end, we monitored the effects of treatment of mice with diesel exhaust, benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP), or a combination of diesel exhaust with either carcinogenic PAH. The applied diesel particulate matter (SRM(1975)) altered the tumor initiating potency of DBP: a statistically significant decrease in overall tumor and carcinoma burden was observed following 25 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with DBP exposure alone. From those mice that were treated at the beginning of the observation period with 2 nmol DBP all survivors developed tumors (9 out of 9 animals, 100%). Among all tumors counted at the end, nine carcinomas were detected and an overall tumor incidence of 2.6 tumors per tumor-bearing animal (TBA) was determined. By contrast, co-treatment of DBP with 50mg SRM(1975) led to a tumor rate of only 66% (19 out of 29 animals), occurrence of only three carcinomas in 29 animals and an overall rate of 2.1 tumors per TBA (P=0.04). In contrast to the results with DBP, the tumor incidence induced by 200 nmol BP was found slightly increased when co-treatment with SRM(1975) occurred (71% vs. 85% after 25 weeks). Despite this difference in tumor incidence, the numbers of carcinomas and tumors per TBA did not differ statistically significant between both treatment groups possibly due to the small size of the BP treatment group. Since bioactivation of DBP, but not BP, predominantly depends on CYP1B1 enzyme activity, SRM(1975) affected PAH-induced carcinogenesis in an antagonistic manner when CYP1B1-mediated bioactivation was required. The explanation most likely lies in the much stronger inhibitory effects of certain PAHs present in diesel exhaust on CYP1B1 compared to CYP1A1. In the present study we also found molecular markers such as highly elevated AKR1C21 and TNFRSF21 gene expression levels in tumor tissue derived from animals co-treated with SRM(1975) plus DBP. Therefore we validate microarray data as a source to uncover transcriptional signatures that may provide insights into molecular pathways affected following exposure to environmental complex mixtures such as diesel exhaust particulates.

Copper delivery to the CNS by CuATSM effectively treats motor neuron disease in SODG93A mice co-expressing the Copper-Chaperone-for-SOD

Over-expression of mutant copper, zinc superoxide dismutase (SOD) in mice induces ALS and has become the most widely used model of neurodegeneration. However, no pharmaceutical agent in 20 years has extended lifespan by more than a few weeks. The Copper-Chaperone-for-SOD (CCS) protein completes the maturation of SOD by inserting copper, but paradoxically human CCS causes mice co-expressing mutant SOD to die within two weeks of birth. Hypothesizing that co-expression of CCS created copper deficiency in spinal cord, we treated these pups with the PET-imaging agent CuATSM, which is known to deliver copper into the CNS within minutes. CuATSM prevented the early mortality of CCSxSOD mice, while markedly increasing Cu, Zn SOD protein in their ventral spinal cord. Remarkably, continued treatment with CuATSM extended the survival of these mice by an average of 18 months. When CuATSM treatment was stopped, these mice developed ALS-related symptoms and died within 3 months. Restoring CuATSM treatment could rescue these mice after they became symptomatic, providing a means to start and stop disease progression. All ALS patients also express human CCS, raising the hope that familial SOD ALS patients could respond to CuATSM treatment similarly to the CCSxSOD mice.

Competitive inhibition of carcinogen‐activating CYP1A1 and CYP1B1 enzymes by a standardized complex mixture of PAH extracted from coal tar

A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, the Standard Reference Material (SRM) 1597, was recently shown to decrease the levels of DNA binding of the 2 strong carcinogens benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) in the human mammary carcinoma‐derived cell line MCF‐7 (Mahadevan et al., Chem Res Toxicol 2005;18:224–231). The present study was designed to further elucidate the biochemical mechanisms involved in this inhibition process. We examined the effects of SRM 1597 on the metabolic activation of BP and DBP toward DNA‐binding derivatives in Chinese hamster cells expressing either human cytochrome P450 (CYP) 1A1 or CYP1B1. SRM 1597 inhibited BP‐DNA adduct formation through the entire exposure time in cells expressing human CYP1A1, while it significantly inhibited adduct formation only up to 48 hr when co‐treated with DBP. Conversely, human CYP1B1‐expressing cells were unable to catalyze PAH‐DNA adduct formation on treatment with SRM 1597 alone, and on co‐treatment with BP or DBP. The data obtained from biochemical experiments revealed that SRM 1597 competitively inhibited the activity of both human enzymes as analyzed by 7‐ethoxyresorufin O‐deethylation assays. While the Michaelis‐Menten constant (KM) was <0.4 μM in the absence of SRM 1597, this value increased up to 1.12 (CYP1A1) or 4.45 μM (CYP1B1) in the presence of 0.1 μg/ml SRM 1597. Hence the inhibitory effects of the complex mixture on human CYP1B1 were much stronger when compared to human CYP1A1. Taken together, the decreases in PAH‐DNA adduct formation on co‐treatment with SRM 1597 revealed inhibitory effects on the CYP enzymes that convert carcinogenic PAH into DNA‐binding metabolites. The implications for the tumorigenicity of complex environmental PAH mixtures are discussed.

The rainbow trout (Oncorhynchus mykiss) tumor model: Recent applications in low-dose exposures to tumor initiators and promoters

The rainbow trout has been utilized as a model for human carcinogenesis for a number of years. Trout are relatively inexpensive to maintain and exhibit (over the 9—12-month tumor assay period) very low spontaneous tumor backgrounds. One of the most powerful applications of this model is the design and conduct of large-scale tumor studies requiring thousands of animals that address statistically challenging questions of dose-response. Two recent examples of such applications include our studies on I3C as a tumor promoter and DBP as a tumor initiator. I3C was shown to promote AFB1- initiated liver cancer at doses near those recommended for supplementation in humans. Further studies are required to determine if the mechanisms responsible for promotion in trout can be extrapolated to humans. In the second example, we report results from the largest animal tumor study ever conducted. A total of 42,000 trout were utilized to measure DBP carcinogenesis down to incidences of 1 in 5,000. The dose response model deviated significantly from linearity although the existence of a threshold could not be statistically established. Extrapolation of the data model predicts a DBP dose producing 1 in 106 cancers that is 1,000-fold higher than predicted by the conservative linear model. If these results can be confirmed with other carcinogens (genotoxic and perhaps nongenotoxic) and other targets, this could have a significant impact on the utilization of animal tumor data in human risk assessment.

Molecular dosimetry in fish : quantitative target organ DNA adduction and hepatocarcinogenicity for four aflatoxins by two exposure routes in rainbow trout

Rainbow trout, a species highly sensitive to aflatoxins, was used to investigate the relative carcinogenicities of four structurally related aflatoxins in terms of their target organ DNA binding characteristics. Tritiated syntheses were carried out, DNA binding dose-response curves were established, and liver DNA binding indices were calculated for the four aflatoxins following a 2-week dietary fry exposure protocol. The results indicated that adduct levels increased linearly with dietary dose concentration, with relative DNA binding indices of 20.7, 20.3, 2.35, and 2.22 x 10(3) (pmoles aflatoxin mg-1 DNA)/(pmoles aflatoxin g-1 diet) for aflatoxin B1 (AFB1), aflatoxicol (AFL), aflatoxin M1 (AFM1), and aflatoxicol M1 (AFLM1), respectively. A similar protocol used over 7200 trout fry averaging 1.2 g initial body weight to establish full carcinogen dose-response curves for each aflatoxin, along with a single-dose estimate of DNA binding index within the tumor study animals. Owing to trout sensitivity a total of 180 micrograms or less of each aflatoxin was required. Data analyzed on logit incidence vs. Ln dose coordinates generated four curves which were modeled as parallel in slope over most or all dose ranges studied. By this analysis, relative tumorigenic potencies were: AFB1 1.00; AFL 0.936; AFM1 0.086; and AFLM1 0.041. When data were plotted as logit incidence vs. Ln adducts (effective dose received), all aflatoxin adducts described the same dose-response curve; that is, they were equally tumorigenic, except those from AFLM1, which were 2-3 fold less potent. Therefore, by these molecular dose studies, differences in tumorigenicity among the four dietary aflatoxins are largely or entirely accounted for by differences in uptake and metabolism leading to DNA adduction, rather than any inherent differences in tumor initiating potency per DNA adduct.