Tissue Organoid Cultures Metabolize Dietary Carcinogens Proficiently and Are Effective Models for DNA Adduct Formation.

Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.

Metabolic Activation of Benzo[a]pyrene by Human Tissue Organoid Cultures.

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.

The Single-Cell Gel Electrophoresis Genotoxin Sensitivity Assay.

The single-cell gel electrophoresis-based genotoxin sensitivity assay (GSA) is an ex vivo approach which enables to study the impact of a variety of dietary factors, occupational exposures, and diseases on the sensitivity of humans towards genotoxic chemicals which cause adverse health effects such as cancer, accelerated aging, and infertility.

Induction of chromosomal damage in exfoliated buccal and nasal cells of road markers.

Road markers are exposed to various chemicals and particles. The aim of this study was to determine whether road worker exposure induceschromosomal damage which is indicative for increased cancer risks. Micronucleus (MN) cytome assays were thus conducted with exfoliated nasal and buccal cells collected from 42 workers and 42 matched controls. The frequencies of MN (reflecting chromosomal aberrations), nuclear buds (NBuds; reflecting gene amplifications) and binucleated cells (BN; reflecting disturbed mitosis) were scored. Further, the rates of nuclear anomalies indicative of acute cytotoxicity (condensed chromatin, karyorrhexis, karyolysis, pyknosis) were evaluated. Data demonstrated marked induction of MN, NBuds, and BN by 1.34-fold, 1.24-fold and 1.14-fold in buccal cells. In nasal cells, only MN frequencies were elevated, 1.23-fold. These effects were paralleled by increased rates of condensed chromatin, karyorrhexis and karyolysis in both cell types. The effects were more pronounced in individuals who had worked for more than 10 years while smoking did not produce synergistic responses. This is the first investigation concerning the induction of genetic damage in road markers and the results are suggestive for enhanced cancer risks. It is conceivable that exposure to silica dust (known to induce cancer and genetic damage) and/or benzoyl peroxide which forms reactive radicals may be associated with the observed genetic damage in road workers. Further investigations of the cancer risks of these workers are warranted.

Methamphetamine ("crystal meth") causes induction of DNA damage and chromosomal aberrations in human derived cells.

Methamphetamine (METH) is a widely consumed psychostimulant drug; its acute toxic effects in brain and liver are well known, furthermore, there is some evidence in regard to its DNA damaging properties in humans. Therefore, we studied the impact of the drug on genomic stability in human derived hepatoma (HepG2) cells, which reflect the activation/detoxification of drugs better than other cell lines. Furthermore, experiments with human buccal derived cells (TR146) were conducted as the drug is consumed orally. Induction of DNA damage in both cell types with doses reflecting the exposure in abusers was found in single cell gel electrophoresis (SCGE) assays (which detect single and double strand breaks as well as apurinic sites). Furthermore, induction of micronuclei (formed as a consequence of structural and numerical chromosomal aberrations) and formation of nuclear buds resulting from gene amplifications was detected. Additional experiments with lesion-specific enzymes showed that the drug causes oxidation of purines and pyrimidines, indicating that its genotoxic effects may be due to oxidation of the DNA. Our findings support the assumption that the drug may cause adverse health effects (such as cancer and infertility) in long-term users which are causally related to DNA damage.

Impact of extended working periods on genomic and telomeric DNA and on inflammatory markers: Results of an intervention study with office workers and carpenters.

Aim of this study was to clarify if extension of the work phase has an impact on DNA- stability, telomere lengths and inflammatory markers. We conducted an intervention trial with office workers (n = 24) and carpenters (n = 10), who changed their working schedule from 8 to 12 h per day over a period of 3 months. The work of both groups involved only moderate physical activity. We found no evidence for induction of double strand breaks (measured in γH2AX assays) and relative telomere lengths (relTL_36B4 and ALB) in lymphocytes in the two study groups. Furthermore, no overall changes of the levels of C-reactive protein (CRP), interleukin-6 (IL-6) and thiobarbituric acid reactive substances (TBARS) in plasma were detected. However, we found in agreement with earlier investigations a moderate (not significant) increase of the CRP levels with age. Furthermore, significant higher CRP concentrations (P = 0.03) were detected in young individuals (21-30 years) as a consequence of the extended working period. Taken together our findings indicate that prolongation of the working hours has no pronounced impact on DNA stability, telomere shortening and inflammatory markers; but the increase of the CRP concentrations in young workers may be indicative for adverse health effects in this subgroup.

Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells.

Some epidemiological studies indicate that the use of mobile phones causes cancer in humans (in particular glioblastomas). It is known that DNA damage plays a key role in malignant transformation; therefore, we investigated the impact of the UMTS signal which is widely used in mobile telecommunications, on DNA stability in ten different human cell lines (six brain derived cell lines, lymphocytes, fibroblasts, liver and buccal tissue derived cells) under conditions relevant for users (SAR 0.25 to 1.00 W/kg). We found no evidence for induction of damage in single cell gel electrophoresis assays when the cells were cultivated with serum. However, clear positive effects were seen in a p53 proficient glioblastoma line (U87) when the cells were grown under serum free conditions, while no effects were found in p53 deficient glioblastoma cells (U251). Further experiments showed that the damage disappears rapidly in U87 and that exposure induced nucleotide excision repair (NER) and does not cause double strand breaks (DSBs). The observation of NER induction is supported by results of a proteome analysis indicating that several proteins involved in NER are up-regulated after exposure to UMTS; additionally, we found limited evidence for the activation of the γ-interferon pathway. The present findings show that the signal causes transient genetic instability in glioma derived cells and activates cellular defense systems.

Gallic Acid Improves Health-Associated Biochemical Parameters and Prevents Oxidative Damage of DNA in Type 2 Diabetes Patients: Results of a Placebo-Controlled Pilot Study.

SCOPE: Oxidative imbalance plays a key role in cancer induction and cardiovascular diseases (CVD) in patients with type 2 diabetes mellitus (T2DM). The aim of this study is to find out if gallic acid (GA) prevents oxidative stress in diabetic patients. Therefore, we investigate its impact on oxidation of DNA bases and on other health-related macromolecules. METHODS AND RESULTS: We perform an intervention study (n = 19) with GA and monitored alterations of the DNA stability in single cell gel electrophoresis (SCGE) assays in lymphocytes. Furthermore, a panel of health-related biomarkers is measured before and after consumption of GA (15 mg p-1  d-1 ) for 7 d. Significant reduction of oxidized purines (by 31%, p < 0.001, effect size 0.404) and pyrimidines (by 2%, p < 0.022, effect size 0.089) is observed in SCGE assays. Furthermore, the plasma concentrations of oxidized-LDL and C-reactive protein are reduced after the intervention by 24% (p = 0.014, effect size 0.384) and 39% (p < 0.001, effect size 0.686), respectively. No alterations of other biomarkers are found. CONCLUSIONS: A small amount of GA (in the range of daily consumption in Central Europe) prevents oxidative DNA damage and reduces markers which reflect inflammation and increased risks of cancer and CVD.

Evaluation of the potential of mobile phone specific electromagnetic fields (UMTS) to produce micronuclei in human glioblastoma cell lines.

Some epidemiological studies indicate that mobile phones cause glioblastomas in humans. Since it is known that genomic instability plays a key role in the etiology of cancer, we investigated the effects of the universal mobile telecommunications system radiofrequency (UMTS-RF) signal, which is used in "smart" phones, on micronucleus (MN) formation and other anomalies such as nuclear buds (NBUDs) and nucleoplasmatic bridges (NPBs). MN are formed by structural and numerical aberrations, NBs reflect gene amplification and NPBs are formed from dicentric chromosomes. The experiments were conducted with human glioblastoma cell lines, which differ in regard to their p53 status, namely U87 (wild-type) and U251 (mutated). The cells were cultivated for 16h in presence and absence of fetal calf serum and exposed to different SAR doses (0.25, 0.50 and 1.00W/kg), which reflect the exposure of humans, in presence and absence of mitomycin C as former studies indicate that RF may cause synergistic effects in combination with this drug. We found no evidence for induction of MN and other anomalies. However, with the highest dose, induction of apoptosis was observed in U251 cells on the basis of the morphological features of the cells. Our findings indicate that the UMTS-RF signal does not cause chromosomal damage in glioblastoma cells; the mechanisms which lead to induction of programmed cell death will be investigated in further studies.

Xanthohumol Prevents DNA Damage by Dietary Carcinogens: Results of a Human Intervention Trial.

Xanthohumol (XN) is a hop flavonoid contained in beers and soft drinks. In vitro and animal studies indicated that XN has DNA and cancer protective properties. To find out if it causes DNA protective effects in humans, an intervention trial was conducted in which the participants (n = 22) consumed a XN containing drink (12 mg XN/P/d). We monitored prevention of DNA damage induced by representatives of major groups of dietary carcinogens [i.e., nitrosodimethylamine (NDMA) benzo(a)pyrene (B(a)P) and the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)]. Lymphocytes were collected before, during, and after the intervention and incubated with the carcinogens and with human liver homogenate (S9). We found substantial reduction of B(a)P and IQ (P < 0.001 for both substances) induced DNA damage after consumption of the beverage; also, with the nitrosamine a moderate, but significant protective effect was found. The results of a follow-up trial (n = 10) with XN pills showed that the effects are caused by the flavonoid and were confirmed in γH2AX experiments. To elucidate the underlying mechanisms we measured several parameters of glutathione related detoxification. We found clear induction of α-GST (by 42.8%, P < 0.05), but no alteration of π-GST. This observation provides a partial explanation for the DNA protective effects and indicates that the flavonoid also protects against other carcinogens that are detoxified by α-GST. Taken together, our findings support the assumption that XN has anticarcinogenic properties in humans. Cancer Prev Res; 10(2); 153-60. ©2016 AACR.

Impact of xanthohumol (a prenylated flavonoid from hops) on DNA stability and other health-related biochemical parameters: Results of human intervention trials.

SCOPE: Xanthohumol (XN) is a hop flavonoid found in beers and refreshment drinks. Results of in vitro and animal studies indicate that it causes beneficial health effects due to DNA protective, anti-inflammatory, antioxidant, and phytoestrogenic properties. Aim of the present study was to find out if XN causes alterations of health-related parameters in humans. METHODS AND RESULTS: The effects of the flavonoid were investigated in a randomized crossover intervention trial (n = 22) in which the participants consumed a XN drink (12 mg XN/P/day). We monitored alterations of the DNA stability in single cell gel electrophoresis assays in lymphocytes and of several health-related biomarkers. A decrease of oxidatively damaged purines and protection toward reactive oxygen species induced DNA damage was found after the consumption of the beverage; also the excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-guanosine in urine was reduced. The assumption that the flavonoid causes DNA protection was confirmed in a randomized follow-up study with pure XN (n = 10) with a parallel design. Other biochemical parameters reflecting the redox- and hormonal status and lipid- and glucose metabolism were not altered after the intervention. CONCLUSION: Taken together, our data indicate that low doses of XN protect humans against oxidative DNA damage.

Genotoxic properties of XLR-11, a widely consumed synthetic cannabinoid, and of the benzoyl indole RCS-4.

Aim of this study was the investigation of the genotoxic properties of XLR-11 [1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone, a widely consumed synthetic cannabinoid (SC), and of the benzoyl indole RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone). We characterized the DNA-damaging properties of these drugs in different experimental systems. No evidence for induction of gene mutations was detected in bacterial (Salmonella/microsome) tests, but clear dose-dependent effects were found in in vitro single cell gel electrophoresis (SCGE) assays with human lymphocytes and with buccal- and lung-derived human cell lines (TR-146 and A-549). These experiments are based on the determination of DNA migration in an electric field and enable the detection of single- and double-strand breaks and apurinic sites. Furthermore, we found that both drugs induce micronuclei which are formed as a consequence of chromosomal aberrations. The lack of effects in SCGE experiments with lesion-specific enzymes (FPG, Endo III) shows that the DNA damage is not caused by formation of oxidatively damaged bases; experiments with liver enzyme homogenates and bovine serum albumin indicate that the drugs are not converted enzymatically to DNA-reactive intermediates. Furthermore, results with buccal- and lung-derived human cells show that gaseous treatment of the cells under conditions which reflect the exposure situation in drug users may cause damage of the genetic material in epithelia of the respiratory tract. Since DNA instability is involved in the etiology of cancer, these findings can be taken as an indication that consumption of the SCs may cause tumors in the respiratory tract of consumers.

Investigations of the genotoxic properties of two synthetic cathinones (3-MMC, 4-MEC) which are used as psychoactive drugs.

Synthetic cathinones (SCAs) are consumed worldwide as psychostimulants and are increasingly marketed as surrogates of classical illicit drugs via the internet. The genotoxic properties of most of these drugs have not been investigated. Results of earlier studies show that amphetamines which are structurally closely related to these compounds cause damage to the genetic material. Therefore, we tested the genotoxic properties of two widely consumed SCAs, namely, 3-MMC (2-(methylamino)-1-(3-methylphenyl) propan-1-one) and 4-MEC (2-(ethylamino)-1-(4-methylphenyl) propan-1-one) in a panel of genotoxicity tests. We found no evidence for induction of gene mutations in Salmonella/microsome assays, but both drugs caused positive results in the single cell gel electrophoresis (SCGE) assay which detects single and double strand breaks of DNA in a human derived buccal cell line (TR146). 3-MMC induced similar effects as 4-MEC and also caused significant induction of micronuclei which are formed as a consequence of structural and chromosomal aberrations. Negative results obtained in SCGE experiments with lesion specific enzymes (FPG and Endo III) show that these drugs do not cause oxidative damage of DNA. However, moderate induction of TBARS (which leads to the formation of DNA-reactive substances) was observed with 4-MEC, indicating that the drug causes lipid peroxidation while no clear effect was detected with 3-MMC. Results obtained with liver homogenate in SCGE-experiments show that phase I enzymes do not lead to the formation of DNA reactive metabolites. Taken together, our findings indicate that consumption of certain SCAs may cause adverse health effects in users as a consequence of damage to the genetic material.

Impact of a synthetic cannabinoid (CP-47,497-C8) on protein expression in human cells: evidence for induction of inflammation and DNA damage.

Synthetic cannabinoids (SCs) are marketed worldwide as legal surrogates for marihuana. In order to predict potential health effects in consumers and to elucidate the underlying mechanisms of action, we investigated the impact of a representative of the cyclohexylphenols, CP47,497-C8, which binds to both cannabinoid receptors, on protein expression patterns, genomic stability and on induction of inflammatory cytokines in human lymphocytes. After treatment of the cells with the drug, we found pronounced up-regulation of a variety of enzymes in nuclear extracts which are involved in lipid metabolism and inflammatory signaling; some of the identified proteins are also involved in the endogenous synthesis of endocannabinoids. The assumption that the drug causes inflammation is further supported by results obtained in additional experiments with cytosols of LPS-stimulated lymphocytes which showed that the SC induces pro-inflammatory cytokines (IL12p40 and IL-6) as well as TNF-α. Furthermore, the proteome analyses revealed that the drug causes down-regulation of proteins which are involved in DNA repair. This observation provides an explanation for the formation of comets which was seen in single-cell gel electrophoresis assays and for the induction of micronuclei (which reflect structural and numerical chromosomal aberrations) by the drug. These effects were seen in experiments with human lymphocytes which were conducted under identical conditions as the proteome analysis. Taken together, the present findings indicate that the drug (and possibly other structurally related SCs) may cause DNA damage and inflammation in directly exposed cells of consumers.

Genotoxic properties of representatives of alkylindazoles and aminoalkyl-indoles which are consumed as synthetic cannabinoids.

Synthetic cannabinoids (SCs) cause similar effects as cannabis and are sold in herbal mixtures. Recent investigations indicate that some of these drugs possess genotoxic properties. Therefore, we tested representatives of two groups, namely, aminoalkylindoles (AM-2201 and UR-144) and 1-alkylindazoles (5F-AKB-48 and AM-2201-IC) in single cell gel electrophoresis and micronucleus (MN) assays with human lymphocytes and in Salmonella/microsome assays. All drugs except AM-2201 caused DNA-migration, the LOELs were between 50 and 75 µM. Furthermore, all SCs caused inhibition of cell division and significant induction of MN which reflect structural and numerical chromosomal aberrations. The LOEL values were 50 µM for UR-144 and 5-AKB-48 and 75 µM for the other drugs. Also the levels of nucleoplasmatic bridges which are formed from dicentric chromosomes were elevated under identical conditions while the frequencies of nuclear buds were not affected. These findings show that representatives of both groups cause chromosomal damage while the negative results in Salmonella assays (in strains TA98, TA100, TA1535, TA1537 and TA102) in absence and presence of metabolic activation indicate that they do not induce gene mutations. Taken together, these findings indicate that SCs may cause adverse health effects in users as a consequence of damage of the genetic material.