Detection of photogenotoxicity in skin and eye in rat with the photo comet assay

Photosensitizing drugs increase the sensitivity of the skin and the eye toward normally harmless sunlight conditions and are known to enhance the induction of skin tumors or severe injuries to the eye. The photogenotoxicity of five common drugs (sparfloxacin, dacarbazine, chlorpromazine and 8-methoxypsoralen, promazine) was investigated in the skin as well as in the retina and cornea of Wistar rats. The compounds were administered once orally by gavage and the resulting DNA damage was analyzed in the newly developed in vivo photo comet assay. All drugs except of promazine were clearly photogenotoxic in the skin. In the cornea sparfloxacin and dacarbazine induced an increased DNA damage following irradiation. A photogenotoxic effect in the retina was observed by sparfloxacin, which is the only compound tested that absorbs wavelengths reaching the retina. The drug concentration analysis revealed that the compounds were distributed into plasma, skin and eye at concentrations, which were photogenotoxic in vitro. Additionally, histopathological analysis showed no relevant alterations or inductions of necrosis, apoptosis or inflammation in the skin or eye. In conclusion, we confirmed the photogenotoxic potential of compounds from different chemical classes in the skin. Moreover, it is the first time that photogenotoxicity has been detected in the retina and cornea in an in vivo study. Based on our results it is concluded that the photo comet assay in rat is an easy and reliable method to elucidate drug induced photogenotoxicity under conditions, which are relevant to human exposure.     

Comprehensive assessment of the photomutagenicity, photogenotoxicity and photo(cyto)toxicity of azulene

The polycyclic aromatic hydrocarbon azulene and its naturally occurring derivative guaiazulene (1,4-dimethyl-7-isopropylazulene) are known to absorb light in the UV-vis region of the spectrum. Both compounds were reported to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Ames test) in strain TA102, and to cause DNA damage in the comet assay in vitro upon exposure to UVA light. In contrast, another study reported a photoprotective effect in vitro of guaiazulene. We present here a comprehensive assessment of the photo(cyto)toxicity (3T3 fibroblast Neutral Red uptake test), the photomutagenicity (Ames test) and photogenotoxicity (comet assay and micronucleus test in L5178Y cells in vitro) of azulene. In the Ames test, the mutagenicity of azulene was assessed in the presence and absence of UV light by use of the Salmonella strains TA102, TA104, TA2638 and E. coli WP2. Azulene was irradiated before being plated with bacteria (pre-irradiation), or concomitantly with the bacteria either after plating or while in suspension. Guaiazulene was included in some of the experiments. Neither in the photo-Ames test nor in the other photogenotoxicity tests, azulene or guaiazulene showed any photomutagenic or photogenotoxic activity. Weak photo(cyto)toxicity (estimate of PIF≥1.67) was observed with azulene in the 3T3 NRU test, the Alamar Blue test and the relative cell count, which may be due to the generation of reactive oxygen species, as reported recently.     

The application of the micronucleus test in Chinese hamster V79 cells to detect drug-induced photogenotoxicity.

Recent reports on the photochemical carcinogenicity and photochemical genotoxicity of fluoroquinolone antibacterials led to an increasing awareness for the need of a standard approach to test for photochemical genotoxicity. In this study the micronucleus test using V79 cells was adapted to photogenotoxicity testing. Results of using different UVA/UVB relationships enabled us to identify a suitable irradiation regimen for the activation of different kinds of photosensitizers. Using this regimen, 8-methoxypsoralen and the fluoroquinolones lomefloxacin, grepafloxacin and Bay Y 3118 were identified to cause micronuclei and toxicity upon photochemical activation. Among the phenothiazines tested, chlorpromazine and 2-chlorophenothiazine, were positive for both endpoints, whereas triflupromazine was only slightly photoclastogenic in the presence of strong phototoxicity. Among the other potential human photosensitizers tested (oxytetracycline, doxycycline, metronidazole, emodin, hypericin, griseofulvin), only hypericin was slightly photogenotoxic. Photochemical toxicity in the absence of photochemical genotoxicity was noted for doxycycline and emodin. With the assay system described, it is possible to determine photochemical toxicity and photochemical genotoxicity concomitantly with sufficient reliability.     

A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue

A quantitative colorimetric assay using the oxidation-reduction indicator Alamar Blue was developed to measure cytotoxicity of compounds against the protozoan parasite Leishmania. Absorbance increased linearly with the plating density of promastigotes of L. major MRHO/IR/76 vaccine strain up to at least 2.5 x 10(6) cells/ml when parasites were incubated for 72 h in the presence of 10% Alamar Blue. The 50% effective dose values of common drugs (amphotericin B, pentostam and paromomycin) obtained by this assay were in the same range as previously determined by other methods. The Alamar Blue assay permits a simple, reproducible and reliable method for screening antileishmanial drugs.     

Prevention of the emergence of drug resistance in bacteria by acridines, phenothiazines, and dibenzocycloheptenes

It has been found that, like Atabrine, the phenothiazine tranquilizers promazine, chlorpromazine, promethazine, levopromethazine, and Stelazine; the antidepressants (dibenz-azepine and dibenzocycloheptene derivatives) Tofranil, Pertofrane, cyclobenzaprine, Elavil, protriptyline, and 3-chlorodibenzocycloheptene; and the acridine derivatives acridine orange, acriflavin, SKF no. 16214-A2, SKF no. 13231-A2, SKF no. 9200, and SKF no. 9836 are all to a greater or lesser extent than Atabrine, effective in preventing the emergence of resistant strains of Staphylococcus aureus and Escherichia coli in the presence of streptomycin, sulfathiazole, or chloramphenicol. The antimalarials chloroquine and hydroxychloroquine were also studied and found to be ineffective. The medical significance of these findings is discussed, as well as the effect of the structural variations of these compounds on their relative activities.     

Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay

The urgent need for new drug development for African trypanosomiasis is widely recognized. This requires reliable and informative high-throughput assays. Currently, drug action is determined with a fluorimetric/colorimetric assay based on the metabolism of the dye Alamar Blue (resazurin) by live cells. However, this assay does not easily distinguish between cell death and growth arrest, or supply information about the rate at which test compounds affect these parameters. We report here an alternative fluorimetric assay, based on the interaction of propidium iodide with DNA, that allows either real-time monitoring of cell viability or the generation of EC₅₀ values at a predetermined time-point. The assay is highly sensitive and fluorescence readings easily correlate to numbers of parasites or DNA content. The EC₅₀ values were highly similar to those obtained with the standard Alamar Blue assay. The procedure lends itself readily to applications in drug development or resistance monitoring.     

Exploration of novel in vitro assays to study drugs against Trichuris spp

Though trichuriasis is a significant public health problem, few effective drugs are available underscoring the need for new drug therapies. For the evaluation of trichuricidal activity of test compounds in vitro an accurate, reliable, sensitive, fast and cheap drug sensitivity assay is essential. The aim of the present investigation was to evaluate the performance of different in vitro drug sensitivity assays in comparison to the standard motility assay. Trichuris muris L4 larvae or adult worms were isolated from the intestinal tract from infected female C57BL/10 mice and incubated in the presence of ivermectin, levamisole and nitazoxanide (200, 100 and 50 μg/ml) for 72 h. The health status of the worms was either evaluated microscopically using a motility scale from 0 to 3 (motility assay), by examination of absorbance or emission in response to metabolic activity (MTT (Thiazolyl Blue Tetrazolium Bromide) and Alamar Blue assay), through analysis of absorbance of an enzyme-substrate reaction (acid phosphatase activity assay), by measuring the noise amplitudes (isothermal microcalorimetry and xCELLigence System) or the heat flow (isothermal microcalorimetry) of T. muris. The Alamar Blue assay, xCELLigence and microcalorimetry compared favorably to the standard motility assay. These three assays precisely determined the trichuricidal activity of the three test drugs. The acid phosphatase and the MTT assays showed a poorer performance than the motility assay. In conclusion, the colorimetric Alamar Blue in vitro assay is a good alternative to the motility assay to study drug effects against T. muris L4 and adults, since it is easy to perform, precise and of low cost.     

The effect on sister-chromatid exchanges of drugs and dyes by intercalation and photoactivation.

Intercalating dyes (acridine orange, proflavin and methylene blue) and drugs (chlorpromazine, promazine and chlorprothixene) were tested for their ability to induce sister-chromatid exchanges (SCEs) with and without photoactivation by visible light. Whereas in the dark all substances tested increased the frequency of SCEs, a superimposed effect of visible light on SCE formation was observed for the acridines proflavin and acridine orange, but not for the pheneothiazine derivatives methylene blue and chlorpromazine. These results are discussed in connection with the known mutagenic effects of these substances and with the factors that may be involved in SCE formation induced by intercalating molecules in the absence and presence of visible light.     

Survival and mutagenic responses of mitomycin C-sensitive mouse lymphoma cell mutants to other DNA cross-linking agents

Mitomycin C-sensitive mutants MCN 151 (complementation group I) and MCE 50 (complementation group II) derived from mouse lymphoma L5178Y cells were found to be also highly sensitive to the lethal effects of other DNA cross-linking agents, such as photoaddition of 8-methoxypsoralen (8-MOP) and cis-diamminedichloroplatinum II (cis-DDP). They were less sensitive to the monofunctional derivative 3-carbethoxypsoralen (3-CPs) and to trans-DDP to trans-DDP than their bifunctional counterparts. Incorporation levels of labeled 8-MOP or 3-CPs in wild-type cells and 2 mutants were almost the same, indicating that the sensitivity is not caused by differential incorporation of the agents. The rates of photoinduced mutations to 6-thioguanine resistance in the mutants, per unit dose of 8-MOP, were about 4 times higher for MCN 151 and 3 times higher for MCE 50 than that in L5178Y cells. However, the rates of induced mutations per viable cells in the mutants were nearly equal to those in wild-type cells. Cross-link repair was compared between mutants and wild-type cells by using the alkaline sucrose-gradient sedimentation technique. The results show that normal cells and both mutants are able to incise the cross-linked DNA, which is the first step of cross-link repair.     

Real-time fluorescence-based detection of furanocoumarin photoadducts of DNA

Real-time fluorescence detection systems were adapted to identify DNA adducts formed by photogenotoxic phytochemicals. Two assays were developed: the first was based on quantitative polymerase chain reaction (qPCR) while the second used thermal denaturation and renaturation (D-R). Both assays employed yeast DNA, the fluorescent dye SYBR Green and a real-time PCR thermocycler. The furanocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), psoralen, angelicin and imperatorin, and the furanochrome khellin, were tested for adduct forming ability with up to 2 h of UVA light exposure (lambda = 320-400 nm). The known bifunctional compounds, 8-MOP, 5-MOP and psoralen, were inferred to form biadducts here based on both D-R and qPCR assays, as expected from previous research. The known monofunctional compound angelicin was used as a negative control and did not form biadducts based on either assay. Two compounds of unknown functional specificity, imperatorin and khellin, were determined to be positive and negative for biadduct activity, respectively. Detection of biadducts with 8-MOP, 5-MOP, psoralen and imperatorin, but not angelicin or khellin, was further verified by temperature gradient gel electrophoresis. The fluorescence methods improve and expand upon existing assays to monitor DNA adducts.     

Mutagenic activity of some coffee flavor ingredients

The mutagenicity of 4 coffee flavor ingredients (chlorogenic acid, caffeic acid, pyrazine, and trigonelline) was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK +/- assay. Two of the compounds, pyrazine and trigonelline, were negative in both assays. The other two compounds, caffeic acid and chlorogenic acid, were positive in the mouse lymphoma assay but negative in the Salmonella assay.     

Bacteriological follow-up of pulmonary tuberculosis treatment: a study with a simple colorimetric assay

The viability of Mycobacterium tuberculosis (MTB) in serial sputum specimens from persistently smear positive patients was evaluated. The assay was based on oxidation-reduction of Alamar Blue and Malachite Green dyes that change their color in response to MTB growth. A total of 280 sputum specimens from 40 persistently smear positive TB patients and 40 sputa from non-tuberculosis patients were digested, decontaminated and examined microscopically. To check the MTB viability, the sediments from decontaminated samples were inoculated into three culture media: Lowenstein-Jensen (LJ) slants, Alamar Blue and Malachite Green culture tubes. We found that out of 280 smear positive specimens, the LJ culture was positive in 124 (44%). The numbers of correctly identified S+/C+ cases by Alamar Blue and Malachite Green were 118 (95%) and 116 (93%), respectively. The mean time required for reporting the positive signal in Alamar Blue culture tubes was 9 versus 11 days by Malachite Green culture tubes. In the standard LJ culture media the average detection time was 27 days (P < 0.05). The sensitivity of LJ was 99%, Alamar Blue 95% and Malachite Green 93%. The specificity was 100%, 92% and 93%, respectively. The oxidation-reduction method is rapid, sensitive and inexpensive in monitoring the treatment response of patients with pulmonary TB. Thus, using this method can be of paramount importance, particularly in resource-constrained areas.     

Inverse correlation between bacterial frameshift mutagenicity and yeast mitochondrial effects of antitumour anilinoacridines

The mutagenicity of a series of derivatives of 9-anilinoacridine, including the clinical antitumour agent amsacrine, has been assessed using a bacterial frameshift tester strain (Salmonella typhimurium TA1537) and a yeast petite colony assay (Saccharomyces cerevisiae 5178B). The results have been compared with microbial mammalian cell cytotoxicity, DNA binding affinity and acridine base strength (pKa). Compounds containing strong electron donor substituents on the acridine ring, and which have a high acridine pKa, show minimal frameshift mutagenicity but are strong inducers of petite yeast mutants. Conversely, some compounds which have a high DNA binding constant but a significant proportion of uncharged form at neutral pH, show high frameshift mutagenicity but minimal induction of petite mutants. It is hypothesised that this inverse relationship arises from the presence of trans-membrane drug transport mechanisms which act to exclude some compounds, particularly strongly basic compounds from the cytoplasm and to concentrate them in mitochondria.     

Photo-chemically induced DNA effects in the comet assay with epidermal cells of SKH-1 mice after a single oral administration of different fluoroquinolones and 8-methoxypsoralen in combination with exposure to UVA

Due to the need for in vivo photo-genotoxicity tests, the in vivo photo-comet assay was established in epidermal cells of the SKH-1 mouse. Groups of 10 male SKH-1 mice each were treated once orally with vehicle only, with three fluoroquinolones (25 mg/kg clinafloxacin, 20 mg/kg lomefloxacin, 200 mg/kg ciprofloxacin) or with 200mg/kg 8-methoxypsoralene (8-MOP). Thirty minutes after treatment half of the mice in each group were exposed to 23.8 J/cm2 UVA. Thereafter the mice were killed and their epidermal cells tested in the alkaline (pH >13) comet assay; at the same time after administration, compound-treated, non-irradiated mice were killed and analysed. A negative control group of ten male SKH-1 mice received the vehicle only; half of these animals were exposed to UVA, half were not. The comet tail lengths of epidermal cells of the mice were statistically significantly increased for all three fluoroquinolones (FQ) tested in combination with UV irradiation. Treatment with 8-methoxypsoralene+UV induced a significant reduction of comet tail length. Tail intensity and tail moment gave essentially the same results after combined exposure (compound+UV). Without irradiation, the tail lengths of controls and compound-treated mice were comparable under the conditions of this study. In contrast, tail intensity and tail moment were increased for all test compounds (including 8-MOP), without irradiation. Irradiated controls had a tail length comparable to non-irradiated controls, while tail intensity and tail moment were clearly increased in irradiated controls. In conclusion: under the present experimental conditions the in vivo photo-comet assay is able to detect photo-chemically induced DNA strand breaks as well as photo-chemically induced DNA cross-links.     

Comparative mutagenicity testing of bropirimine, 1. Induction of chromosome aberrations in CHO cells is not reflected in induction of mutation at the TK locus of L5178Y cells

Bropirimine (U-54,461) is a novel compound which is being developed as a biological response modifier for use in treatment of neoplastic and viral disease. Compounds of this type exert their therapeutic effects by immuno-stimulation or other non-cytotoxic mechanisms. The purpose of the experiments described in this paper was to evaluate the hazard potential of this drug. Bropirimine was previously reported to be negative in the Ames Salmonella assay (Aaron et al., 1989a) and the in vitro UDS assay (Aaron et al., 1989b). In experiments reported here positive response was observed in a test for clastogenicity in vitro in CHO cells, but bropirimine was negative in the L5178Y mouse lymphoma TK^+/− assay. A subsequent experiment demonstrated the ability of bropirimine to induce HPRT mutations in CHO cells. Interestingly, evidence for induction of chromosome aberrations in the L5178Y cells by bropirimine was also obtained. While the reason for the apparent insensitivity of the L5178Y TK^+/− assay to bropirimine is unexplained by the experiments, it is clear that at high dose bropirimine is capable of clastogenesis in both CHO and L5178Y cells and can give rise to gene mutations in CHO cells but apparently not in L5178Y cells.     

Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds?

Today reconstructed skin models that simulate human skin, such as Episkin, are widely used for safety or efficacy pre-screening. Moreover, they are of growing interest for regulatory purposes in the framework of alternatives to animal testing. In order to reduce and eventually replace results of in vivo genotoxicity testing with in vitro data, there is a need to develop new complementary biological models and methods with improved ability to predict genotoxic risk. This can be achieved if these new assays do take into account exposure conditions that are more relevant than in the current test systems. In an attempt to meet this challenge, two new applications using a human reconstructed skin model for in vitro genotoxicity assessment are proposed. The skin is the target organ for dermally exposed compounds or environmental stress. Although attempts have been made to develop genotoxicity test procedures in vivo on mouse skin, human reconstructed skin models have not been used for in vitro genotoxicity testing so far, although they present clear advantages over mouse skin for human risk prediction. This paper presents the results of the development of a specific protocol allowing to perform the comet assay, a genotoxicity test procedure, on reconstructed skin. The comet assay was conducted after treatment of Episkin with UV, Lomefloxacin and UV or 4-nitroquinoline-N-oxide (4NQO). Treatment with the sunscreen Mexoryl was able to reduce the extent of comet signal. A second approach to use reconstructed epidermis in genotoxicity assays is also proposed. Indeed, the skin is a biologically active barrier driving the response to exposure to chemical agents and their possible metabolites. A specific co-culture system (Figure 1) using Episkin to perform the regular micronucleus assay is presented. Micronucleus induction in L5178Y cells cultured underneath Episkin was assessed after treatment of the reconstructed epidermis with mitomycin C, cyclophosphamide or apigenin. This second way of using human reconstructed skin for genotoxicity testing aims at improving the relevance of exposure conditions in in vitro genotoxicity assays for dermally applied compounds.     

An evaluation of protein assays for quantitative determination of drugs

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.     

The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.     

Evaluation of mutagenic effects of hyperbaric oxygen (HBO) in vitro. II. Induction of oxidative DNA damage and mutations in the mouse lymphoma assay

We recently showed that treatment of V79 cells with hyperbaric oxygen (HBO) efficiently induced DNA effects in the comet assay and chromosomal damage in the micronucleus test (MNT), but did not lead to gene mutations at the hprt locus. Using the comet assay in conjunction with bacterial formamidopyrimidine DNA glycosylase (FPG protein), we now provide indirect evidence that the same treatment leads to the induction of 8-oxoguanine, a premutagenic oxidative DNA base modification in V79 and mouse lymphoma (L5178Y) cells. We also demonstrate that HBO efficiently induces mutations in the mouse lymphoma assay (MLA). Exposure of L5178Y cells to HBO (98% O(2); 3bar) for 2h caused a clear mutagenic effect in the MLA, which was further enhanced after a 3h exposure. As this mutagenic effect was solely due to the strong increase of small colony (SC) mutants, we suggest that HBO causes mutations by induction of chromosomal alterations. Molecular characterization of induced SC mutants by loss of heterozygosity (LOH) analysis showed an extensive loss of functional tk sequences similar to the pattern found in spontaneous SC mutants. This finding confirmed that the majority of HBO-induced mutants is actually produced by a clastogenic mechanism. The induction of point mutations as a consequence of induced oxidative DNA base damage seems to be of minor importance.     

Use of the alkaline comet assay for the detection of transplacental genotoxins in newborn mice

Several lines of evidence show that in utero exposure to different toxicants has greater consequences than their exposure during adult life. This may be due to involvement of critical developmental stages, physiological immaturity and the long later-life span over which disease may initiate, develop and progress. The in vivo alkaline comet (single-cell gel electrophoresis) assay has been favoured by the scientific community for the evaluation of genotoxins. The objective of this study was to demonstrate the suitability of alkaline comet assay in detecting transplacental genotoxins using newborn mice. Here, we report the successful use of the comet assay in detecting multi-organ genotoxicity of known transplacental genotoxins in newborn mice. Three well known transplacental genotoxic agents, cyclophosphamide (CP), mitomycin-C (MMC) and zidovudine (AZT) were tested in pregnant Swiss mice. These compounds were administered in the late gestational period (16-20th days of pregnancy) and the comet assay was performed with lymphocytes, bone marrow, liver and kidney cells of newborn mice. Significant DNA damage was observed in all the tissues with tested transplacental genotoxins. The results of the comet assay were confirmed by the micronucleus (MN) assay of the peripheral blood of newborn mice. The results of this study provide sufficient evidence that the comet assay can be applied successfully for the detection of transplacental genotoxins in newborn mice.