Evaluation of Pharmaceuticals for DNA Damage in the Chicken Egg Genotoxicity Assay (CEGA).

DNA damage is an established initiating event in the mutagenicity and carcinogenicity of genotoxic chemicals. Accordingly, assessment of this endpoint is critical for chemicals which are being developed for use in humans. To assess the ability of the Chicken Egg Genotoxicity Assay (CEGA) to detect genotoxic pharmaceuticals, a set of 23 compounds with different pharmacological and reported genotoxic effects was tested for the potential to produce nuclear DNA adducts and strand breaks in the embryo-fetal livers using the 32P-nucleotide postlabeling (NPL) and comet assays, respectively. Due to high toxicity, two aneugens, colchicine and vinblastine, and an autophagy inhibitor, hydroxychloroquine, could not be evaluated. Out of the 20 remaining pharmaceuticals, 10 including estrogen modulators, diethylstilbestrol and tamoxifen, antineoplastics cyclophosphamide, etoposide, and mitomycin C, antifungal griseofulvin, local anesthetics lidocaine and prilocaine, and antihistamines diphenhydramine and doxylamine, yielded clear positive outcomes in at least one of the assays. The antihypertensive vasodilator hydralazine and antineoplastics streptozotocin and teniposide, produced only DNA strand breaks, which were not dose-dependent, and thus, the results with these 3 pharmaceuticals were considered equivocal. No DNA damage was detected for 7 compounds, including the purine antagonist 6-thioguanine, antipyretic analgesics acetaminophen and phenacetin, antibiotic ciprofloxacin, antilipidemic clofibrate, anti-inflammatory ibuprofen, and sedative phenobarbital. However, low solubility of these compounds limited dosages tested in CEGA. Overall, results in CEGA were largely in concordance with the outcomes in other systems in vitro and in vivo, indicating that CEGA provides reliable detection of DNA damaging activity of genotoxic compounds. Further evaluations with a broader set of compounds would support this conclusion.

High-Performance Breaking and Intelligent of Miniature Circuit Breakers.

The exploitation and utilization of clean energy such as wind and photovoltaic power plays an important role in the reduction in carbon emissions to achieve the goal of "emission peak and carbon neutral", but such a quantity of clean energy accessing the electric system will foster the transition of the electric power system structure. The intelligentization of power equipment will be an inevitable trend of development. High breaking performance, remote control and a digital detection platform of miniature circuit breaker, a protective equipment of a power distribution system, have also been inevitable requirements of the power Iot system. Based on the above, this paper studies three aspects: high-performance AC and DC general switching technology, remote control technology and operation status' digital monitoring. A new DC non-polar breaking technology is proposed, which improves the short circuit breaking ability. An experimental prototype using the above techniques was fabricated and passed the DC 1000 V/10 kA short-circuit breaking test. On the basis of the above, an intelligent circuit breaker is developed, which contains multiple functions: remote switching, real-time temperature detection, energy metering and fault warning. Moreover, a software for digital condition monitoring and remote control is developed. This work has certain theoretical and practical significance for the development of the power Internet of things.

A Single-Phase High-Impedance Ground Faulty Feeder Detection Method for Small Resistance to Ground Systems Based on Current-Voltage Phase Difference.

At present, the small resistance to ground system (SRGS) is mainly protected by fixed-time zero-sequence overcurrent protection, but its ability to detect transition resistance is only about 100 Ω, which is unable to detect single-phase high resistance grounding fault (SPHIF). This paper analyzes the zero-sequence characteristics of SPHIF for SRGS and proposes a SPHIF feeder detection method that uses the current-voltage phase difference. The proposed method is as follows: first, the zero-sequence current phase of each feeder is calculated. Second, the phase voltage root mean square (RMS) value is used to determine the fault phase and obtain its initial phase as the reference value. The introduction of the initial phase of the fault phase voltage can highlight the fault characteristics and improve the sensitivity and reliability of feeder detection, and then CVPD is the difference between each feeder ZSC phase and the reference value. Finally, the magnitude of CVPD is judged. If the CVPD of a particular feeder meets the condition, the feeder is detected as the faulted feeder. Combining the theoretical and practical constraints, the specific adjustment principle and feeder detection logic are given. A large number of simulations show that the proposed method can be successfully detected under the conditions of 5000 Ω transition resistance, -1 dB noise interference, and 40% data missing. Compared with existing methods, the proposed method uses phase voltages that are easy to measure to construct SPHIF feeder detection criteria, without adding additional measurement and communication devices, and can quickly achieve local isolation of SPHIF with better sensitivity, reliability, and immunity to interference.

Analysis of the Influence of the Breaking Radiation Magnetic Field of a 10 kV Intelligent Circuit Breaker on an Electronic Transformer.

The development of the smart grid requires the distribution switch to not be limited to the original breaking function. More functional requirements lead to more complex switch structures, especially the intelligent processing unit on the secondary side. A technology called primary and secondary integration optimizes the structure of the switch, which greatly increases the intelligence level of the switch, but also has disadvantages. The secondary intelligent unit is arranged close to the primary high-voltage electromagnetic environment, and the distribution switch is prone to failure due to electromagnetic interference. In order to explore the influence of electromagnetic interference on it, a transient electromagnetic interference simulation test platform was built for a 10 kV intelligent distribution switch based on the principle of spherical gap arc discharge, and the interference signal of the intelligent distribution switch was measured; the law of the spatial magnetic field near the electronic transformer is mainly studied in this paper. The shielding effectiveness of the distribution terminal of the switch was analyzed, and the interference of the power line of the sensor merging unit circuit board was calculated. The results show that the electronic transformer may have serious faults under continuous strong transient electromagnetic interference. The electromagnetic transient simulation test system studied in this paper can evaluate the anti strong electromagnetic interference ability of the electronic transformer.

Electricity Consumption Forecasting using Support Vector Regression with the Mixture Maximum Correntropy Criterion.

The electricity consumption forecasting (ECF) technology plays a crucial role in the electricity market. The support vector regression (SVR) is a nonlinear prediction model that can be used for ECF. The electricity consumption (EC) data are usually nonlinear and non-Gaussian and present outliers. The traditional SVR with the mean-square error (MSE), however, is insensitive to outliers and cannot correctly represent the statistical information of errors in non-Gaussian situations. To address this problem, a novel robust forecasting method is developed in this work by using the mixture maximum correntropy criterion (MMCC). The MMCC, as a novel cost function of information theoretic, can be used to solve non-Gaussian signal processing; therefore, in the original SVR, the MSE is replaced by the MMCC to develop a novel robust SVR method (called MMCCSVR) for ECF. Besides, the factors influencing users' EC are investigated by a data statistical analysis method. We find that the historical temperature and historical EC are the main factors affecting future EC, and thus these two factors are used as the input in the proposed model. Finally, real EC data from a shopping mall in Guangzhou, China, are utilized to test the proposed ECF method. The forecasting results show that the proposed ECF method can effectively improve the accuracy of ECF compared with the traditional SVR and other forecasting algorithms.

Electricity Consumption Forecasting Scheme via Improved LSSVM with Maximum Correntropy Criterion.

In recent years, with the deepening of China's electricity sales side reform and electricity market opening up gradually, the forecasting of electricity consumption (FoEC) becomes an extremely important technique for the electricity market. At present, how to forecast the electricity accurately and make an evaluation of results scientifically are still key research topics. In this paper, we propose a novel prediction scheme based on the least-square support vector machine (LSSVM) model with a maximum correntropy criterion (MCC) to forecast the electricity consumption (EC). Firstly, the electricity characteristics of various industries are analyzed to determine the factors that mainly affect the changes in electricity, such as the gross domestic product (GDP), temperature, and so on. Secondly, according to the statistics of the status quo of the small sample data, the LSSVM model is employed as the prediction model. In order to optimize the parameters of the LSSVM model, we further use the local similarity function MCC as the evaluation criterion. Thirdly, we employ the K-fold cross-validation and grid searching methods to improve the learning ability. In the experiments, we have used the EC data of Shaanxi Province in China to evaluate the proposed prediction scheme, and the results show that the proposed prediction scheme outperforms the method based on the traditional LSSVM model.

Sparse-Aware Bias-Compensated Adaptive Filtering Algorithms Using the Maximum Correntropy Criterion for Sparse System Identification with Noisy Input.

To address the sparse system identification problem under noisy input and non-Gaussian output measurement noise, two novel types of sparse bias-compensated normalized maximum correntropy criterion algorithms are developed, which are capable of eliminating the impact of non-Gaussian measurement noise and noisy input. The first is developed by using the correntropy-induced metric as the sparsity penalty constraint, which is a smoothed approximation of the ℓ 0 norm. The second is designed using the proportionate update scheme, which facilitates the close tracking of system parameter change. Simulation results confirm that the proposed algorithms can effectively improve the identification performance compared with other algorithms presented in the literature for the sparse system identification problem.

Assessment of DNA Binding and Oxidative DNA Damage by Acrylonitrile in Two Rat Target Tissues of Carcinogenicity: Implications for the Mechanism of Action.

Exposure to acrylonitrile induces formation of tumors at multiple sites in rats, with females being more sensitive. The present study assessed possible mechanisms of acrylonitrile tumorigenicity, covalent DNA binding, DNA breakage, and oxidative DNA damage, in two target tissues, the brain and Zymbal's glands, of sensitive female Fischer (F344) and Sprague-Dawley (SD) rats. One group received acrylonitrile in drinking water at 100 ppm for 28 days. Two other groups were administered either acrylonitrile in drinking water at 100 ppm or drinking water alone for 27 days, followed by a single oral gavage dose of 11 mg/kg bw 14C-acrylonitrile on day 28. A positive control group received a single dose of 5 mg/kg bw of 7-14C-benzo[a]pyrene, on day 27 following the administration of drinking water for 26 days. Using liquid scintillation counting, no association of radiolabeled acrylonitrile with brain DNA was found. In accelerator mass spectrometry analysis, the association of 14C of acrylonitrile with DNA in brains was detected and was similar in both strains, which may reflect acrylonitrile binding to protein as well as to DNA. Nucleotide 32P-postlabeling assay analysis of brain samples from rats of both strains yielded no evidence of acrylonitrile DNA adducts. Negative conventional comet assay results indicate the absence of direct DNA strand breaks in the brain and Zymbal's gland in both strains of rats dosed with acrylonitrile. In both rat strains, positive results in an enhanced comet assay were found only in brain samples digested with formamidopyrimidine-DNA glycosylase but not with human 8-hydroxyguanine-DNA glycosylase, indicating possible oxidative DNA damage, other than 8-oxodG formation. In conclusion, definitive evidence of DNA binding of acrylonitrile in the brain and Zymbal's gland was not obtained under the test conditions. A role for oxidative stress in tumorigenesis in the brain but not Zymbal's gland may exist.

Sex differences in DNA damage produced by the carcinogen 2-acetylaminofluorene in cultured human hepatocytes compared to rat liver and cultured rat hepatocytes.

Male rats are more susceptible to the induction of liver cancer by the aromatic amine 2-acetylaminofluorene (AAF) than are females. To assess the basis for this difference and to determine whether sex differences in susceptibility to AAF are present in human liver cells, the DNA reactivity of AAF was measured in livers of male and female Sprague-Dawley (SD) rats and in cultured SD rat and human hepatocytes of both sexes. In livers of rats administered oral doses of AAF, the total levels of adducts measured by nucleotide postlabelling at up to 8 weeks were about twofold greater in males than in females. Similarly, the level of AAF-DNA adducts formed in cultured male rat hepatocytes dosed with AAF was about twofold greater than in female rat hepatocytes. Also, the level of DNA repair synthesis was about threefold greater in AAF-dosed cultured male rat hepatocytes compared with female, indicating that the greater adduct levels in males was not due to lesser repair. In contrast, in cultured human hepatocytes of both sexes, AAF produced similar levels of adducts and DNA repair synthesis, which were intermediate between those produced in male and female rat hepatocytes. Thus, the greater susceptibility of male rats to AAF hepatocarcinogenicity is due at least in part to greater bioactivation and formation of AAF-DNA adducts in hepatocytes. Moreover, the data from human hepatocytes suggest that human liver could be less susceptible than male rat liver to the carcinogenic effects of aromatic amine carcinogens of the AAF type.

GRAS determination scientific procedures and possible alternatives.

The use of a food substance is Generally Recognized as Safe (GRAS) through scientific procedures or experience based on common use in food. The pivotal data used for GRAS determination must be of common knowledge and should include evidence for safety under the conditions of intended use of the substance. Such evidence includes data on the identity and specifications of the substance, its properties of absorption, distribution, metabolism and excretion, and depending on the level of concern, data on genotoxicity, acute and subchronic toxicity, reproductive and developmental toxicity and carcinogenicity. Several alternative procedures can be used as the replacement for standard scientific procedures in order to improve the GRAS process.

Assessment of no-observed-effect-levels for DNA adducts formation by genotoxic carcinogens in fetal turkey livers.

For genotoxic carcinogens, covalent binding to DNA is a critical initiating event in tumorigenesis. The present research investigated dose-effect relationships of three genotoxic carcinogens representing different structural classes, 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P) and quinoline (QUI), to assess the existence of no-observed-effect-levels (NOELs) for the formation of DNA adducts. Carcinogens were administered into the air sac of fertilized turkey eggs over wide dose ranges in three daily injections on days 22 to 24 of incubation. DNA adducts were measured in the fetal turkey livers by the 32P-nucleotide postlabeling (NPL) assay. B[a]P and QUI produced DNA adducts in a dosage-related manner and exhibited NOELs at 0.65 and 0.35 mg/kg bw/day, respectively. In contrast, 2-AAF formed DNA adducts at all tested dosages down to 0.005 mg/kg bw/day. Benchmark dose (BMD) analysis identified the potencies of 2-AAF and QUI to be similar, while B[a]P was the least potent compound. Overall, findings in fetal turkey livers demonstrated that exposure levels to genotoxic compounds that do not result in DNA adducts can exist but are not evident with all carcinogens of this type. The use of mechanistic dose-effect studies for genotoxic endpoints can provide critical information for prioritization of concerns for risk assessment.

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds in embryo-fetal chicken livers.

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds were assessed in embryo-fetal chicken livers. Formation of DNA adducts and strand breaks were measured using the nucleotide 32P-postlabelling (NPL) and comet assays, respectively. Unsubstituted monocyclic benzene, polycyclic fused ring compound naphthalene, covalently connected polycyclic ring compound biphenyl, and heterocyclic ring compound fluorene did not produce DNA damage. Amino-substituted monocyclic compounds, aniline and p-phenylenediamine, as well as polycyclic 1-naphthylamine were also negative. In contrast, carcinogenic monocyclic methyl-substituted anilines: o-toluidine, 2,6-xylidine, 3,4-dimethylaniline, 4-chloro-o-toluidine; 2 methoxy-substituted methylaniline: p-cresidine; 2,4 and 2,6 diamino- or dinitro- substituted toluenes all produced DNA damage. Genotoxic polycyclic amino-substituted 2-naphthylamine, 4-aminobiphenyl, benzidine, methyl-substituted 3,2'-dimethyl-4-aminobiphenyl and 4-dimethylaminoazobenzene as well as amino- and nitro- fluorenes substituted at the 1 or 2 positions also were positive in at least one of the assays. Overall, the DNA damaging activity of cyclic compounds in embryo-fetal chicken livers reflected the type and position of the substitution on the aromatic ring. Additionally, substituted polycyclic compounds exhibited higher DNA-damaging potency compared to monocyclic chemicals. These results are congruent with in vivo findings in other species, establishing chicken eggs as a reliable system for structure-activity assessment of members of groups of related chemicals.

Assessment and characterization of DNA adducts produced by alkenylbenzenes in fetal turkey and chicken livers.

Formation of DNA adducts by five alkenylbenzenes, safrole, methyl eugenol, eugenol, and asarone with either α- or ß-conformation, was analyzed in fetal avian livers in two in ovo models. DNA reactivity of the carcinogens safrole and methyl eugenol was previously demonstrated in the turkey egg model, whereas non-genotoxic eugenol was negative. In the current study, alkenylbenzenes were also tested in the chicken egg model. Injections with alkenylbenzenes were administered to fertilized turkey or chicken eggs for three consecutive days. Three hours after the last injection, liver samples were evaluated for DNA adduct formation using the 32P-nucleotide postlabeling assay. DNA samples from turkey livers were also analyzed for adducts using mass spectrometry. In both species, genotoxic alkenylbenzenes safrole, methyl eugenol, α- and ß-asarone produced DNA adducts, the presence and nature of which, with exception of safrole, were confirmed by mass spectrometry, validating the sensitivity of the 32P-postlabeling assay. Overall, the results of testing were congruent between fetal turkey and chicken livers, confirming that these organisms can be used interchangeably. Moreover, data obtained in both models is comparable to genotoxicity findings in other species, supporting the usefulness of avian models for the assessment of genotoxicity as a potential alternative to animal models.

Assessment of the medicines lidocaine, prilocaine, and their metabolites, 2,6-dimethylaniline and 2-methylaniline, for DNA adduct formation in rat tissues.

The local anesthetics lidocaine (lido) and prilocaine (prilo) are metabolized to their constituent aromatic amines 2,6-dimethylaniline (DMA, 2,6-xylidine) and 2-methylaniline (MA, o-toluidine), respectively, which are both tumorigenic in rats. The capacity of lido and prilo to form DNA adducts was assessed in major target tissues for aromatic amines in male F344 rats in comparison to equimolar doses of DMA and MA using the (32)P-postlabeling assay. Direct reaction of putative DNA-reactive metabolites N-hydroxy-DMA and N-hydroxy-MA with isolated DNA yielded reference adducts. Rats were dosed by p.o. gavage with 0.5 mmol/kg b.wt. of each test substance or the vehicle either once or daily for 7 days. After repeat administrations of either prilo or lido, DNA adducts were detected in the liver and nasal mucosa. Urinary bladder DNA adducts were detected only in lido and DMA repeat dosed rats. Groups dosed with DMA or MA showed adducts in both single- and multiple-dose groups, except for the single-dose DMA liver and urinary bladder samples, which were below the level of detection. No DNA adducts were detected in any of the white blood cell samples under either dosing regimen. The lido- and prilo-DNA adducts detected were chromatographically indistinguishable from those formed either in DMA- or MA-dosed rats, respectively, or by chemical reaction of the corresponding N-hydroxy derivatives with DNA. Thus, lido and prilo can generate DNA adducts in rats via their aromatic amine metabolites, although at lower levels than equal molar quantities of their amine metabolites.

In ovo testing of flavor and fragrance materials in Turkey Egg Genotoxicity Assay (TEGA), comparison of results to in vitro and in vivo data.

Genotoxicity of flavor and fragrance materials was assessed in Turkey Egg Genotoxicity Assay (TEGA) using 32P-nucleotide postlabeling (NPL) and comet assays to detect hepatic DNA adducts and strand breaks. Twenty materials having results in GADD45a-Gluc 'BlueScreen HC' genotoxicity assay, and standard in vitro and in vivo tests, were selected to evaluate the accuracy of TEGA. Quinoline (QUI) and 2-acetylaminofluorene (AAF) served as positive comparators. Two materials, p-tert-butyldihydrocinnamaldehyde (BDHCA) and methyl eugenol (MEU) produced DNA adducts. BDHCA, p-t-butyl-α-methylhydrocinnamic aldehyde (BMHCA), trans-2-hexenal (HEX) and maltol (MAL) produced DNA strand breaks. Fifteen other materials were negative in both assays. Based on reports of oxidative DNA damage induction by MAL and 4-hydroxy-2.5-dimethyl-3(2H) furanone (HDMF), modified comet assays were conducted. Positive comet findings for MAL were not confirmed, and only equivocal evidence of oxidative damage was found. Accordingly, MAL was judged to have equivocal genotoxicity in TEGA. HDMF was positive in modified comet assay, indicating an ability to produce oxidative DNA damage. TEGA showed modest concordance with results in regulatory in vitro assays. Findings in TEGA, with few exceptions, were concordant with the results of in vivo genotoxicity and carcinogenicity testing. Thus, TEGA is an attractive alternative model for the assessment of genotoxic potential of chemicals in vivo.

Expression of Genes Encoding for Xenobiotic Metabolism After Exposure to Dialkylnitrosamines in the Chicken Egg Genotoxicity Alternative Model.

The Chicken Egg Genotoxicity Assay (CEGA) demonstrated responsiveness to various DNA-reactive chemicals requiring metabolic activation, which implies broad bioactivation capability. To assess potential metabolic competence, expression profiles of metabolic genes in the embryo-chicken fetal liver were determined using microarray technology. Fertilized chicken eggs were injected under the CEGA protocol with vehicle (deionized water [DW]), the activation-dependent carcinogens, diethylnitrosamine (DEN), and N-nitrosodiethanolamine (NDELA) at doses producing no effect on survival. Previously in CEGA, DEN produced DNA damage, whereas NDELA did not. Expressions of 463 genes known to encode for phase I and II of endo- and xenobiotic metabolism were detected on the array. DW did not affect the expression of the selected genes, deregulating less than 1% of them. In contrast, DEN at 2 mg/egg and NDELA at 4 mg/egg produced significant transcriptomic alterations, up-regulating up to 41% and down-regulating over 31% of studied genes. Both nitrosamines modulated the majority of the genes in a similar manner, sharing 64 up-regulated and 93 down-regulated genes with respect to control group, indicating similarity in the regulation of their metabolism by avian liver. Differences in gene expression between DEN and NDELA were documented for several phase I CYP 450 genes that are responsible for nitrosamine biotransformation, as well as for phase II genes that regulate detoxication reactions. These findings could underlie the difference in genotoxicity of DEN and NDELA in CEGA. In conclusion, the analysis of gene expression profiles in embryo-chicken fetal liver dosed with dialkylnitrosamines demonstrated that avian species possess a complex array of inducible genes coding for biotransformation.

Inhibition by acetaminophen of neoplastic initiation elicited in rat liver by the DNA-reactive hepatocarcinogen N-acetyl-2-aminofluorene.

Acetaminophen, a monocyclic phenolic compound and analgesic, when fed at 8900 p.p.m. in the diet, was reported to inhibit the hepatocarcinogenicity in rats of the aromatic amine proximate carcinogen N-hydroxy-N-acetyl-2-aminofluorene. To elucidate the mechanism(s) of this anticarcinogenicity, the present study examined whether acetaminophen at lower doses has the ability to inhibit the initiating effects in the rat liver of the precursor hepatocarcinogen N-acetyl-2-aminofluorene. Male F344 rats were allocated to six groups, which were maintained under reverse light cycle conditions to assure acetaminophen ingestion at the time of N-acetyl-2-aminofluorene administration during the dark phase, which was imposed from 07.00 to 19.00 h. Group 1 served as vehicle control (0.5% carboxymethylcellulose) for N-acetyl-2-aminofluorene, which was administered intragastrically 3 days per week at 2.6 mg/kg for 8 weeks (group 4) to achieve initiation. Acetaminophen was given in the diet either alone at 2400 or 4800 p.p.m. for 9 weeks (groups 2 and 3), or with N-acetyl-2-aminofluorene (groups 5 and 6), starting 1 week before N-acetyl-2-aminofluorene administration. Acetaminophen blood levels were about 1 and 4 microg/ml at the two dietary concentrations. N-acetyl-2-aminofluorene induced hepatocellular preneoplastic lesions measured as hepatocellular altered foci expressing glutathione S-transferase-P, reflecting initiation. Induced foci were reduced with administration of both concentrations of acetaminophen. Acetaminophen by itself produced no DNA adducts nor did it alter the high formation of N-acetyl-2-aminofluorene-DNA adducts, about 200 in 10 nucleotides, measured by nucleotide postlabeling. Acetaminophen did not affect background liver cell proliferation, but significantly reduced N-acetyl-2-aminofluorene-induced increased proliferation measured by proliferating cell nuclear antigen immunostaining. Thus, acetaminophen effectively protected hepatocytes from the initiating effects of N-acetyl-2-aminofluorene, possibly through a cytoprotective effect resulting from slowing the rate of induced cell turnover.

Inhibition by dietary hydroquinone of acetylaminofluorene induction of initiation of rat liver carcinogenesis.

Monocyclic phenolics (MPs) occur widely in foods, both naturally and as synthetic antioxidant additives. Several have been shown to inhibit the carcinogenicity of a variety of genotoxic carcinogens in various tissues. Hydroquinone (HQ), one of the simplest of the MPs, which occurs naturally as the glucose conjugate arbutin, was studied for its ability, at low dietary levels, to inhibit the initiating effects in the rat liver of the DNA-reactive carcinogen 2-acetylaminofluorene (AAF). Male Fischer 344 rats (F344), 8 weeks old at the start of the study, were allocated to six groups. HQ was fed daily ad libitum in PMI certified diet at either 0.05% (approximately 25 mg/kg bw/d) or 0.2% (approximately 100 mg/kg bw/d) for 13 weeks, starting one week before AAF administration was initiated, and at the same doses to two groups not receiving AAF. AAF was given intragastrically three times a week for 12 weeks at doses of 3mg/kg bw in 0.5% carboxymethyl cellulose (CMC) to a basal diet group and two of the groups receiving HQ in the diet. Vehicle controls were fed basal diet and administered 0.5% CMC intragastrically three times a week. The rats were observed daily and body weights were taken before initial dosing and at weekly intervals thereafter. Body weight gain over time, terminal body weights and absolute (mg) and relative liver weights (relative to body weight) were measured. At the end of the study (13 weeks), DNA adducts ((32)P-postlabeling), cell proliferation (PCNA immunohistochemistry) and preneoplastic hepatocellular altered foci (HAF) (glutathione S-transferase-placental type immuno-histochemistry) were measured. No significant differences were observed in body weight gains or liver weights. AAF produced liver DNA adducts and at the low dose of HQ adduct levels were 90% of that for AAF alone, whereas at the high dose adducts were reduced by 33% (p<0.05). AAF exposure yielded about a 50% increase in hepatocellular proliferation and both HQ doses reduced the AAF-induced increases in proliferation by about 25%. Likewise, the AAF-induced GST-P-positive HAF per cm(2) of liver tissue were decreased by both doses of HQ by about 50%. Thus, under the conditions of this experiment, HQ at both 0.05% and 0.2% in the diet diminished AAF-induced cancer initiating effects in rat liver.

Structure-Activity Relationships for DNA Damage by Alkenylbenzenes in Turkey Egg Fetal Liver.

Certain alkenylbenzenes (AB), flavoring chemicals naturally occurring in spices and herbs, are established to be cytotoxic and hepatocarcinogenic in rodents. The purpose of the present study was to determine the DNA damaging potential of key representatives of this class using the Turkey Egg Genotoxicity Assay. Medium white turkey eggs with 22- to 24-day-old fetuses received three injections of nine AB with different carcinogenic potentials: safrole (1, 2 mg/egg), methyl eugenol (2, 4 mg/egg), estragole (20, 40 mg/egg), myristicin (25, 50 mg/egg), elemicin (20, 50 mg/egg), anethole (5, 10 mg/egg), methyl isoeugenol (40, 80 mg/egg), eugenol (1, 2.5 mg/egg), and isoeugenol (1, 4 mg/egg). Three hours after the last injection, fetal livers were harvested for measurement of DNA strand breaks, using the comet assay and DNA adducts formation, using the nucleotide(3) (2)P-postlabeling assay. Estragole, myristicin, and elemicin induced DNA stand breaks. These compounds as well as safrole, methyl eugenol and anethole, at the highest doses tested, induced DNA adduct formation. Methyl isoeugenol, eugenol, and isoeugenol did not induce genotoxicity. The genotoxic AB all had the structural features of either a double bond in the alkenyl side chain at the terminal 2',3'-position, favorable to formation of proximate carcinogenic 1'-hydroxymetabolite or terminal epoxide, or the absence of a free phenolic hydroxyl group crucial for formation of a nontoxic glucuronide conjugate. In contrast, methyl isoeugenol, eugenol and isoeugenol, which were nongenotoxic, possessed chemical features, unfavorable to activation.

Significantly enhanced tumor cellular and lysosomal hydroxychloroquine delivery by smart liposomes for optimal autophagy inhibition and improved antitumor efficiency with liposomal doxorubicin.

Hydroxychloroquine (HCQ) inhibits autophagy and therefore can sensitize some cancer cells to chemotherapy, but the high doses required limit its clinical use. Here we show that loading HCQ into liposomes (HCQ/Lip) decorated with a pH-sensitive TH-RGD targeting peptide (HCQ/Lip-TR) can concentrate HCQ in B16F10 tumor cells and lysosomes. HCQ/Lip-TR was efficiently internalized as a result of its ability to bind ITGAV-ITGB3/integrin αvß3 receptors highly expressed on the tumor cell surface and to undergo charge reversal from anionic at pH 7.4 to cationic at pH 6.5. Studies in vitro at pH 6.5 showed that the intracellular HCQ concentration was 35.68-fold higher, and lysosomal HCQ concentration 32.22-fold higher, after treating cultures with HCQ/Lip-TR than after treating them with free HCQ. The corresponding enhancements observed in mice bearing B16F10 tumors were 15.16-fold within tumor cells and 14.10-fold within lysosomes. HCQ/Lip-TR was associated with milder anemia and milder myosuppressive reductions in white blood cell and platelet counts than free HCQ, as well as less accumulation in the small intestine, which may reduce risk of intestinal side effects. In addition, co-delivering HCQ/Lip-TR with either free doxorubicin (DOX) or liposomal DOX improved the ability of DOX to inhibit tumor growth. Biochemical, electron microscopy and immunofluorescence experiments confirmed that HCQ/Lip-TR blocked autophagic flux in tumor cells. Our results suggest that loading HCQ into Lip-TR liposomes may increase the effective concentration of the inhibitor in tumor cells, allowing less toxic doses to be used.