Translating dosimetry of Dibenzo[def,p]chrysene (DBC) and metabolites across dose and species using physiologically based pharmacokinetic (PBPK) modeling.

Dibenzo[def,p]chrysene (DBC) is an environmental polycyclic aromatic hydrocarbon (PAH) that causes tumors in mice and has been classified as a probable human carcinogen by the International Agency for Research on Cancer. Animal toxicity studies often utilize higher doses than are found in relevant human exposures. Additionally, like many PAHs, DBC requires metabolic bioactivation to form the ultimate toxicant, and species differences in DBC and DBC metabolite metabolism have been observed. To understand the implications of dose and species differences, a physiologically based pharmacokinetic model (PBPK) for DBC and major metabolites was developed in mice and humans. Metabolism parameters used in the model were obtained from experimental in vitro metabolism assays using mice and human hepatic microsomes. PBPK model simulations were evaluated against mice dosed with 15 mg/kg DBC by oral gavage and human volunteers orally microdosed with 29 ng of DBC. DBC and its primary metabolite DBC-11,12-diol were measured in blood of mice and humans, while in urine, the majority of DBC metabolites were obeserved as conjugated DBC-11,12-diol, conjugated DBC tetrols, and unconjugated DBC tetrols. The PBPK model was able to predict the time course concentrations of DBC, DBC-11,12-diol, and other DBC metabolites in blood and urine of human volunteers and mice with reasonable accuracy. Agreement between model simulations and measured pharmacokinetic data in mice and human studies demonstrate the success and versatility of our model for interspecies extrapolation and applicability for different doses. Furthermore, our simulations show that internal dose metrics used for risk assessment do not necessarily scale allometrically, and that PBPK modeling provides a reliable approach to appropriately account for interspecies differences in metabolism and physiology.

2-Amino-3-Methylimidazo[4,5-f]quinoline Triggering Liver Damage by Inhibiting Autophagy and Inducing Endoplasmic Reticulum Stress in Zebrafish (Danio rerio).

It is important to note that 2-Amino-3-methylimidazole[4,5-f]quinoline (IQ) is one of the most common heterocyclic amines (HCAs), which is a class of mutagenic/carcinogenic harmful compounds mainly found in high-protein thermal processed foods and contaminated environments. However, the pre-carcinogenic toxicity of IQ to the liver and its mechanism are poorly understood, further research is needed. In light of this, we exposed zebrafish to IQ (0, 8, 80, and 800 ng/mL) for 35 days, followed by comprehensive experimental studies. Histopathological and ultrastructural analysis showed that hepatocytes were damaged. TUNEL results showed that IQ induced apoptosis of liver cells, the expression of apoptosis factor gene was significantly increased, and the expression of Bcl-2 protein was significantly decreased. In addition, upregulated expression of the 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) and endoplasmic reticulum stress (ERS)-related factors transcription levels were elevated obviously, suggesting that IQ induced ERS. Decreased protein expression of autophagy-related 5 (Atg5)-Atg12, Beclin1, and LC3-II, increased protein expression of p62, and autophagy-related factors transcription levels were significantly decreased, suggesting that IQ inhibited autophagy. Overall, our research showed that the potential harm of IQ to the liver before the occurrence of liver cancer was related to ERS and its mediated autophagy and apoptosis pathways.

Parasympathetic, but not sympathetic denervation, suppressed colorectal cancer progression.

Disruption in the nerve-tumor interaction is now considered as a possible anticancer strategy for treating various cancer types, particularly colorectal cancer. However, the underlying mechanisms are not still fully understood. Therefore, the present study aimed to evaluate the effects of sympathetic and parasympathetic denervation on the inhibition of colorectal cancer progression in early and late phases and assess the involvement of nerve growth factor in denervation mediated anticancer effects. One-hundred and fifty male Wistar rats were assigned into 15 groups. Seven groups comprising the control group, 1,2-dimethylhydrazine (DMH) group, sympathetic denervation group (celiac-mesenteric ganglionectomy and guanethidine sulphate administration), parasympathetic denervation group (vagotomy and atropine administration), and combination group were used in the early-stage protocol. For the late-stage protocol, eight groups comprising the control, DMH, surgical and pharmacological sympathetic and parasympathetic denervation groups, combination group, and 5-flourouracil group were considered. After 8 weeks, sympathetic and parasympathetic denervation significantly reduced ACF numbers in rats receiving DMH. On the other hand, in the late stages, parasympathetic but not sympathetic denervation resulted in significant reductions in tumor incidence, tumor volume and weight, cell proliferation (indicated by reduced immunostaining of PCNA and ki-67), and angiogenesis (indicated by reduced immunostaining of CD31 and VEGF expression levels), and downregulated NGF, ß2 adrenergic, and M3 receptors. It can be concluded that parasympathetic denervation may be of high importance in colon carcinogenesis and suggested as a possible therapeutic modality in late stages of colorectal cancer.

Repeated exposure of the oral mucosa over 12 months with cold plasma is not carcinogenic in mice.

Peri-implantitis may result in the loss of dental implants. Cold atmospheric pressure plasma (CAP) was suggested to promote re-osseointegration, decrease antimicrobial burden, and support wound healing. However, the long-term risk assessment of CAP treatment in the oral cavity has not been addressed. Treatment with two different CAP devices was compared against UV radiation, carcinogen administration, and untreated conditions over 12 months. Histological analysis of 406 animals revealed that repeated CAP exposure did not foster non-invasive lesions or squamous cell carcinoma (SCCs). Carcinogen administration promoted non-invasive lesions and SCCs. Molecular analysis by a qPCR screening of 144 transcripts revealed distinct inflammatory profiles associated with each treatment regimen. Interestingly, CAP treatment of carcinogen-challenged mucosa did not promote but instead left unchanged or reduced the proportion of non-invasive lesions and SCC formation. In conclusion, repeated CAP exposure of murine oral mucosa was well tolerated, and carcinogenic effects did not occur, motivating CAP applications in patients for dental and implant treatments in the future.

Lung metabolome of 1,3-butadiene exposed Collaborative Cross mice reflects metabolic phenotype of human lung cancer.

1,3-Butadiene (BD) exposure is known to cause numerous adverse health effects, including cancer, in animals and humans. BD is metabolized to reactive epoxide intermediates, which are genotoxic, but it is not well know what other effects BD has on cellular metabolism. We examined the effects of exposure to BD on the mouse lung metabolome in the genetically heterogeneous collaborative cross outbred mouse model. Mice were exposed to 3 concentra-tions of BD for 10 days (2, 20, and 200 ppm), and lung tissues were analyzed using high-resolution mass spectrometry-based metabolomics. As compared to controls (0 ppm BD), BD had extensive effects on lung metabolism at all concentrations of exposure, including the lowest concentration of 2 ppm, as reflected by reprogramming of multiple metabolic pathways. Metabolites participating in glycolysis and the tricarboxylic acid cycle were elevated, with 8 out of 10 metabolites demonstrating a 2 to 8-fold increase, including the oncometabolite fumarate. Fatty acid levels, sphingosine, and sphinganine were decreased (2 to 8-fold), and fatty acyl-CoAs were significantly increased (16 to 31-fold), suggesting adjustments in lipid metabolism. Furthermore, metabolites involved in basic amino acid metabolism, steroid hormone metabolism, and nucleic acid metabolism were significantly altered. Overall, these changes mirror the metabolic alterations found in lung cancer cells, suggesting that very low doses of BD induce metabolic adaptations that may prevent or promote adverse health effects such as tumor formation.

Prolonged sub-lethal exposure to galaxolide (HHCB) and tonalide (AHTN) promotes the metastatic potential of glioblastoma tumor spheroids.

Galaxolide and tonalide are well-known polycyclic musks whose intensive use without limitations in numerous cleaning, hygiene, and personal care products has resulted in widespread direct human exposure via absorption, inhalation, and oral ingestion. Latest data shows that long-term, low-dose exposure to toxic chemicals can induce unpredictable harmful effects in a variety of living systems, however, interactions between synthetic musks and brain tumours remain largely unexplored. Glioblastoma (GB) accounts for nearly half of all tumours of the central nervous system and is characterized by very poor prognosis. The aims of this study were (1) to investigate the potential effect of long-term (20-generation) single and combined application of galaxolide and tonalide at sub-lethal doses (5-2.5 u M) on the angiogenesis, invasion, and migration of human U87 cells or tumour spheroids, and (2) to explore the underlying molecular mechanisms. Random amplified polymorphic DNA assays revealed significant DNA damage and increased total mutation load in galaxolide- and/or tonalide-treated U87 cells. In those same groups, we also detected remarkable tumour spheroid invasion and up-regulation of both HIF1-α/VEGF/MMP9 and IL6/JAK2/STAT3 signals, known to have important roles in hypoxia-related angiogenesis and/or proliferation. Prolonged musk treatment further altered angio-miRNA expression in a manner consistent with poor prognosis in GB. We also detected significant over-expression of the genes Slug, Snail, ZEB1, and Vimentin, which are biomarkers of epithelial to mesenchymal transition. In addition, matrigel, transwell, and wound healing assays clearly showed that long-term sub-lethal exposure to galaxolide and/or tonalide induced invasion and migration proposing a high metastatic potential. Our results suggest that assessing expression of HIF-1a, VEGF, STAT3, and the miR-17-92 cluster in biopsy samples of GB patients who have a history of possible long-term exposure to galaxolide or tonalide could be beneficial for deciding a therapy regime. Additionally, we recommend that extensively-used hygiene and cleaning materials be selected from synthetic musk-free products, especially when used in palliative care processes for GB patients.

Genotoxicity evaluation using primary hepatocytes isolated from rhesus macaque (Macaca mulatta).

Non-human primates (NHPs) have played a vital role in fundamental, pre-clinical, and translational studies because of their high physiological and genetic similarity to humans. Here, we report a method to isolate primary hepatocytes from the livers of rhesus macaques (Macaca mulatta) after in situ whole liver perfusion. Isolated primary macaque hepatocytes (PMHs) were treated with various compounds known to have different pathways of genotoxicity/carcinogenicity and the resulting DNA damage was evaluated using the high-throughput CometChip assay. The comet data were quantified using benchmark dose (BMD) modeling and the BMD50 values for treatments of PMHs were compared with those generated from primary human hepatocytes (PHHs) in our previous study (Seo et al. Arch Toxicol 2020, 2207-2224). The results showed that despite varying CYP450 enzyme activities, PMHs had the same sensitivity and specificity as PHHs in detecting four indirect-acting (i.e., requiring metabolic activation) and seven direct-acting genotoxicants/carcinogens, as well as five non-carcinogens that are negative or equivocal for genotoxicity in vivo. The BMD50 estimates and their confidence intervals revealed species differences for DNA damage potency, especially for direct-acting compounds. The present study provides a practical method for maximizing the use of animal tissues by isolating primary hepatocytes from NHPs. Our data support the use of PMHs as a reliable surrogate of PHHs for evaluating the genotoxic hazards of chemical substances for humans.

Study on the potential nephrotoxicity and mutagenicity of aristolochic acid IVa and its mechanism.

Previous reports demonstrated that aristolochic acids (AAs) exposure-induced nephrotoxicity, mutations, and tumorigenesis are mainly due to aristolochic acid I (AAI). Notably, the chemical structure of aristolochic acid IVa (AAIVa), which exists at higher levels in many Aristolochiaceae herbs, is extremely similar to AAI. In lack of toxicological data, it is unknown whether AAIVa exposure leads to aristolochic acid nephropathy (AAN), mutations, and tumorigenesis as of AAI. To answer these questions, mice were administered AAIVa by single or repeated long-term gavage, while AAI was used as a positive control. We found that single gavage of 40 mg/kg of AAIVa exhibited no obvious toxicity. Also, there were no tumors or death in mice administrated with 1 and 10 mg/kg of AAIVa for 6 months followed by a 12-month recovery time. There were no noteworthy alterations in gene mutation frequency in the kidney, liver, and stomach between the AAIVa and control mice. Fascinatingly, AA-associated mutational signatures, adenine-to-thymine (A>T) transversions, were absent in AAIVa-treated mice. Nonetheless, 10 mg/kg of AAIVa triggered lymphocytic infiltration and slight fibrous hyperplasia in the kidney at the 6th month; however, these were alleviated at the 12th and 18th months. On the contrary, AAI (positive control) caused severe diffuse fibrosis, tubular atrophy, necrosis, tumors in the forestomach and kidney, and death after the 6th month. It seems that long-term AAIVa exposure induced mild renal lesions could be due to the activation of the canonical or noncanonical transforming growth factor-ß (TGFß) pathway. Overall, these findings suggest that the mutagenicity and carcinogenic risk of AAIVa are very low.

Aristolochic acid IVa forms DNA adducts in vitro but is non-genotoxic in vivo.

Aristolochic acids (AAs) are a family of natural compounds with AA I and AA II being known carcinogens, whose bioactivation causes DNA adducts formation. However, other congeners have rarely been investigated. This study aimed to investigate genotoxicity of AA IVa, which differs from AA I by a hydroxyl group, abundant in Aristolochiaceae plants. AA IVa reacted with 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) to form three dA and five dG adducts as identified by high-resolution mass spectrometry, among which two dA and three dG adducts were detected in reactions of AA IVa with calf thymus DNA (CT DNA). However, no DNA adducts were detected in the kidney, liver, and forestomach of orally dosed mice at 40 mg/kg/day for 2 days, and bone marrow micronucleus assay also yielded negative results. Pharmacokinetic analyses of metabolites in plasma indicated that AA IVa was mainly O-demethylated to produce a metabolite with two hydroxyl groups, probably facilitating its excretion. Meanwhile, no reduced metabolites were detected. The competitive reaction of AA I and AA IVa with CT DNA, with adducts levels varying with pH of reaction revealed that AA IVa was significantly less reactive than AA I, probably by hydroxyl deprotonation of AA IVa, which was explained by theoretical calculations for reaction barriers, energy levels of the molecular orbits, and charges at the reaction sites. In brief, although it could form DNA adducts in vitro, AA IVa was non-genotoxic in vivo, which was attributed to its low reactivity and biotransformation into an easily excreted metabolite rather than bioactivation.

Global Analysis of Protein Expression in A549 Cells After Prolonged Nicotine Exposure by Using Label-free Quantification.

BACKGROUND/AIM: Lung cancer is the leading cause of cancer death worldwide. Cigarette smoke is the most important risk factor for cancer development. Growing evidence indicates that prolonged nicotine exposure is a potential factor associated with tumorigenesis. Here, the effect of prolonged nicotine exposure on A549 cells was investigated, using label-free quantitative proteomics. MATERIALS AND METHODS: Selection of an invasive subpopulation from the A549 cell line was performed to reveal the differential expression of proteins in relation to prolonged nicotine exposure, using Boyden chamber assays in combination with a proteomics approach. RESULTS: One hundred proteins from the NicoA549-L5 subline showed significant change in expression compared to those from the A549-L5 subline and their A549 parental cell line. Heat shock protein, protein disulfide isomerase A3, profilin-1 and legumain were expressed at higher levels in A549 cells after prolonged nicotine exposure. CONCLUSION: These aberrant proteins might serve as novel cancer biomarkers for cigarette smokers.

Chromosome aberrations induced by the non-mutagenic carcinogen acetamide involve in rat hepatocarcinogenesis through micronucleus formation in hepatocytes.

Chromosome aberrations (CAs), i.e. changes in chromosome number or structure, are known to cause chromosome rearrangements and subsequently tumorigenesis. However, the involvement of CAs in chemical-induced carcinogenesis is unclear. In the current study, we aimed to clarify the possible involvement of CAs in chemical carcinogenesis using a rat model with the non-mutagenic hepatocarcinogen acetamide. In an in vivo micronucleus (MN) test, acetamide was revealed to induce CAs specifically in rat liver at carcinogenic doses. Acetamide also induced centromere-positive large MN (LMN) in hepatocytes. Immunohistochemical and electron microscopic analyses of the LMN, which can be histopathologically detected as basophilic cytoplasmic inclusion, revealed abnormal expression of nuclear envelope proteins, increased heterochromatinization, and massive DNA damage. These molecular pathological features in LMN progressed with acetamide exposure in a time-dependent manner, implying that LMN formation can lead to chromosome rearrangements. Overall, these data suggested that CAs induced by acetamide play a pivotal role in acetamide-induced hepatocarcinogenesis in rats and that CAs can cause chemical carcinogenesis in animals via MN formation.

Categorizing the characteristics of human carcinogens: a need for specificity.

The International Agency for Research on Cancer (IARC) has recently proposed employing "ten key characteristics of human carcinogens" (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

A systematic approach to evaluate plausible modes of actions for mouse lung tumors in mice exposed to 4-methylimidozole.

The National Toxicology Program (NTP) reported that chronic dietary exposure to 4-methylimidazole (4-MeI) increased the incidence of lung adenomas/carcinomas beyond the normally high spontaneous rate in B6C3F1 mice. To examine plausible modes of action (MoAs) for mouse lung tumors (MLTs) upon exposure to high levels of 4-MeI, and their relevance in assessing human risk, a systematic approach was used to identify and evaluate mechanistic data (in vitro and in vivo) in the primary and secondary literature, along with high-throughput screening assay data. Study quality, relevance, and activity of mechanistic data identified across the evidence-base were organized according to key characteristics of carcinogens (KCCs) to identify potential key events in known or novel MLT MoAs. Integration of these evidence streams provided confirmation that 4-MeI lacks genotoxic and cytotoxic activity with some evidence to support a lack of mitogenic activity. Further evaluation of contextual and chemical-specific characteristics of 4-MeI was consequently undertaken. Due to lack of genotoxicity, along with transcriptomic and histopathological lung changes up to 28 and 90 days of exposure, the collective evidence suggests MLTs observed following exposure to high levels of 4-MeI develop at a late stage in the mouse chronic bioassay, albeit the exact MoA remains unclear.

Cytotoxicity and genotoxicity of the carcinogen aristolochic acid I (AA-I) in human bladder RT4 cells.

Aristolochic acid (AA-I) induces upper urothelial tract cancer (UUTC) and bladder cancer (BC) in humans. AA-I forms the 7-(2'-deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) adduct, which induces multiple A:T-to-T:A transversion mutations in TP53 of AA-I exposed UTUC patients. This mutation is rarely reported in TP53 of other transitional cell carcinomas and thus recognized as an AA-I mutational signature. A:T-to-T:A transversion mutations were recently detected in bladder tumors of patients in Asia with known AA-I-exposure, implying that AA-I contributes to BC. Mechanistic studies on AA-I genotoxicity have not been reported in human bladder. In this study, we examined AA-I DNA adduct formation and mechanisms of toxicity in the human RT4 bladder cell line. The biological potencies of AA-I were compared to 4-aminobiphenyl, a recognized human bladder carcinogen, and several structurally related carcinogenic heterocyclic aromatic amines (HAA), which are present in urine of smokers and omnivores. AA-I (0.05-10 µM) induced a concentration- and time-dependent cytotoxicity. AA-I (100 nM) DNA adduct formation occurred at over a thousand higher levels than the principal DNA adducts formed with 4-ABP or HAAs (1 µM). dA-AL-I adduct formation was detected down to a 1 nM concentration. Studies with selective chemical inhibitors provided evidence that NQO1 is the major enzyme involved in AA-I bio-activation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bio-activation or detoxification of AA-I. AA-I DNA damage also induced genotoxic stress leading to p53-dependent apoptosis. These biochemical data support the human mutation data and a role for AA-I in BC.

Tumour necrosis factor-α (TNF-α) enhances dietary carcinogen-induced DNA damage in colorectal cancer epithelial cells through activation of JNK signaling pathway.

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazol [4,5-b] pyridine (PhIP) present in cooked meat are pro-carcinogens and considered to be potential risk factors for CRC. Their carcinogenic and mutagenic effects require metabolic activation primarily by cytochrome P450 1 family enzymes (CYPs); the expression of these enzymes can be modulated by aryl hydrocarbon receptor (AhR) activation and the tumour microenvironment, involving mediators of inflammation. In this study, we hypothesized that tumour necrosis factor-α (TNF-α), a key mediator of inflammation, modulates BaP- and PhIP-induced DNA damage in colon cancer epithelial cells. Importantly, we observed that TNF-α alone (0.1-100 pg/ml) induced DNA damage (micronuclei formation) in HCT-116 cells and co-treatment of TNF-α with BaP or PhIP showed higher levels of DNA damage compared to the individual single treatments. TNF-α alone or in combination with BaP or PhIP did not affect the expression levels of CYP1A1 and CYP1B1 (target genes of AhR signaling pathways). The DNA damage induced by TNF-α was elevated in p53 null HTC-116 cells compared to wild type cells, suggesting that TNF-α-induced DNA damage is suppressed by functional p53. In contrast, p53 status failed to affect BaP and PhIP induced micronucleus frequency. Furthermore, JNK and NF-κB signaling pathway were activated by TNF-α treatment but only inhibition of JNK significantly reduced TNF-α-induced DNA damage. Collectively, these findings suggest that TNF-α induced DNA damage involves JNK signaling pathway rather than AhR and NF-κB pathways in colon cancer epithelial cells.

vHTS and 3D-QSAR for the Identification of Novel Phyto-inhibitors of Farnesyltransferase: Validation of Ascorbic Acid inhibition of Farnesyltransferase in an Animal Model of Breast Cancer.

Farnesyltransferase (FTase) is a zinc enzyme that has been the subject of attention in anti-cancer research over the past. In this study, phytochemicals from Curcuma longa L., Taraxacum officinale, and Spondias mombin plants were screened for their inhibitory potentials on the human farnesyltransferase. A three-dimensional quantitative structure-activity relationship (3D-QSAR) model for the inhibition of farnesyltransferase was generated and the inhibition of farnesyltransferase by the hit, ascorbic acid was validated in an animal model of breast cancer. The lead compound, ascorbic acid makes extensive hydrogen bond interactions with key residues, lys-353, tyr-300, gly-290, leu-290 within the active site of farnesyltransferase. It downregulated the expression of FNTA mRNA in an animal model of breast cancer. The 3D-QSAR generated herein is robust, thoroughly validated, and should be employed in the pipelining of novel farnesyltransferase inhibitors. Ascorbic acid demonstrates its anticancer potentials through the inhibition of farnesyltransferase.

A Method to Pre-Screen Rat Mammary Gland Whole-Mounts Prior To RNAscope.

RNAscope is a quantitative in situ gene expression measurement technique that preserves the spatial aspect of intact tissue; thus, allowing for comparison of specific cell populations and morphologies. Reliable and accurate measurement of gene expression in tissue is dependent on preserving RNA integrity and the quantitative nature of RNAscope. The purpose of this study was to determine if the quantitative nature of RNAscope was retained following processing and carmine staining of mammary gland whole-mounts, which are commonly used to identify lesions, such as hyperplasia and ductal carcinoma in situ (DCIS). We were concerned that handling and procedures required to visualize microscopic disease lesions might compromise RNA integrity and the robustness of RNAscope. No effect on the quantitative abilities of RNAscope was detected when mammary gland whole-mounts were pre-screened for lesions of interest prior to RNAscope. This was determined in comparison to tissue that had been formalin-fixed and paraffin embedded (FFPE) immediately after collection. The ability to pre-screen whole-mounts allowed unpalpable diseased lesions to be identified without labor-intensive serial sectioning of tissue samples to find diseased tissue. This method is applicable to evaluate mammary gland whole-mounts during normal mammary gland development, function, and disease progression.

Thresholds for carcinogens.

Current regulatory cancer risk assessment principles and practices assume a linear dose-response relationship-the linear no-threshold (LNT) model-that theoretically estimates cancer risks occurring following low doses of carcinogens by linearly extrapolating downward from experimentally determined risks at high doses. The two-year rodent bioassays serve as experimental vehicles to determine the high-dose cancer risks in animals and then to predict, by extrapolation, the number of carcinogen-induced tumors (tumor incidence) that will arise during the lifespans of humans who are exposed to environmental carcinogens at doses typically orders of magnitude below those applied in the rodent assays. An integrated toxicological analysis is conducted herein to reconsider an alternative and once-promising approach, tumor latency, for estimating carcinogen-induced cancer risks at low doses. Tumor latency measures time-to-tumor following exposure to a carcinogen, instead of tumor incidence. Evidence for and against the concept of carcinogen-induced tumor latency is presented, discussed, and then examined with respect to its relationship to dose, dose rates, and the dose-related concepts of initiation, tumor promotion, tumor regression, tumor incidence, and hormesis. Considerable experimental evidence indicates: (1) tumor latency (time-to-tumor) is inversely related to the dose of carcinogens and (2) lower doses of carcinogens display quantifiably discrete latency thresholds below which the promotion and, consequently, the progression and growth of tumors are delayed or prevented during a normal lifespan. Besides reconciling well with the concept of tumor promotion, such latency thresholds also reconcile favorably with the existence of thresholds for tumor incidence, the stochastic processes of tumor initiation, and the compensatory repair mechanisms of hormesis. Most importantly, this analysis and the arguments presented herein provide sound theoretical, experimental, and mechanistic rationales for rethinking the foundational premises of low-dose linearity and updating the current practices of cancer risk assessment to include the concept of carcinogen thresholds.

Coexposure to Inhaled Aldehydes or Carbon Dioxide Enhances the Carcinogenic Properties of the Tobacco-Specific Nitrosamine 4-Methylnitrosamino-1-(3-pyridyl)-1-butanone in the A/J Mouse Lung.

Tobacco smoke is a complex mixture of chemicals, many of which are toxic and carcinogenic. Hazard assessments of tobacco smoke exposure have predominantly focused on either single chemical exposures or the more complex mixtures of tobacco smoke or its fractions. There are fewer studies exploring interactions between specific tobacco smoke chemicals. Aldehydes such as formaldehyde and acetaldehyde were hypothesized to enhance the carcinogenic properties of the human carcinogen, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) through a variety of mechanisms. This hypothesis was tested in the established NNK-induced A/J mouse lung tumor model. A/J mice were exposed to NNK (intraperitoneal injection, 0, 2.5, or 7.5 µmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 17 ppmv) for 3 h in a nose-only inhalation chamber, and lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had more adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase the severity of NNK-induced lung adenomas. The aldehyde coexposure did not affect the levels of NNK-derived DNA adduct levels. Similar studies tested the ability of a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure to influence lung adenoma formation by NNK. While carbon dioxide alone was not carcinogenic, it significantly increased the number of NNK-derived lung adenomas without affecting NNK-derived DNA damage. These studies indicate that the chemicals in tobacco smoke work together to form a potent lung carcinogenic mixture.

Palmitoyl piperidinopiperidine, a novel derivative of 10­hydroxy­2­decenoic acid, as a potent and selective anticancer agent against human colon carcinoma cell lines.

The present study, to the best of our knowledge, is the first systematic study of the inhibitory effects of palmitoyl piperidinopiperidine (PPI; Japan Patent no. 5597427), on colon carcinogenesis. PPI exhibited marked growth inhibitory activity in several human colon carcinoma cell lines, with IC50 values of approximately 0.5­2.2 µM. In silico docking analysis indicated that PPI could bind to the SH2 domain of signal transducer and activator of transcription 3 (STAT3). PPI markedly inhibited the transcriptional activity of the SW837 cell line. Flowcytometric analysis demonstrated that PPI induced an increase in the number of cells in the G1 phase of the cell cycle, and induced sub­G1 fractions of cells at a higher concentration level of PPI. In the HT29 and SW837 cells, western blot analyses exhibited that in whole cell lysates, PPI induced a marked decrease in the expression levels of p­STAT3, but not in the levels of STAT3 in these cells. PPI also induced a marked decrease in the expression levels of both STAT3 and p­STAT3 in the chromatin fraction. In addition, PPI affected the protein expression levels of cyclin D1, p53, Bcl­2, Bcl­xL and vascular endothelial growth factor (VEGF). In the HT29 cells, PPI induced a marked and dose­dependent increase in the expression levels of Bax, cleaved caspase­3, cleaved caspase­7, cleaved caspase­8, cleaved caspase­9 and cleaved poly (ADP­ribose) polymerase (PARP). In animal model systems, PPI inhibited the growth of implanted carcinoma cells, and also induced a significant decrease in the multiplicity of colonic aberrant crypt foci. In addition, a marked and dose­dependent inhibition of angiogenesis of the chick chorioallantoic membrane was observed. As regards the possible molecular mechanisms, it is suggested that the inhibition of STAT3 by PPI may affect the function of molecules that are related to apoptosis, angiogenesis and cell cycle progression, eventually contributing to the PPI­induced growth inhibitory effects.