The Effectiveness of Therapeutic Vaccines for the Treatment of Cervical Intraepithelial Neoplasia 3: A Systematic Review and Meta-Analysis.

Cervical cancer (CC) is a disease that affects many women worldwide, especially in low-income countries. The human papilloma virus (HPV) is the main causative agent of this disease, with the E6 and E7 oncoproteins being responsible for the development and maintenance of transformed status. In addition, HPV is also responsible for the appearance of cervical intraepithelial neoplasia (CIN), a pre-neoplastic condition burdened by very high costs for its screening and therapy. So far, only prophylactic vaccines have been approved by regulatory agencies as a means of CC prevention. However, these vaccines cannot treat HPV-positive women. A search was conducted in several databases (PubMed, Scopus, Web of Science, and ClinicalTrials.gov) to systematically identify clinical trials involving therapeutic vaccines against CIN 3. Histopathological regression data, immunological parameters, safety, DNA clearance, and vaccine efficacy were considered from each selected study, and from the 102 articles found, 8 were selected based on the defined inclusion criteria. Histopathological regression from CIN 3 to CIN < 1 was 22.1% (95% CI: 0.627−0.967; p-value = 0.024), showing a vaccine efficacy of 23.6% (95% CI; 0.666−0.876; p-value < 0.001). DNA clearance was assessed, and the risk of persistent HPV DNA was 23.2% (95% CI: 0.667−0.885; p-value < 0.001). Regarding immunological parameters, immune responses by specific T-HPV cells were more likely in vaccinated women (95% CI: 1.245−9.162; p-value = 0.017). In short, these studies favored the vaccine group over the placebo group. This work indicated that therapeutic vaccines are efficient in the treatment of CIN 3, even after accounting for publication bias.

Metabolic Profiling of Inga Species with Antitumor Activity.

This work evaluated the metabolic profiling of Inga species with antitumor potential. In addition, we described the antigenotoxicity of polyphenols isolated from I. laurina and a proteomic approach using HepG2 cells after treatment with these metabolites. The in vitro cytotoxic activity against HepG2, HT-29 and T98G cancer cell lines was investigated. The assessment of genotoxic damage was carried out through the comet assay. The ethanolic extract from I. laurina seeds was subjected to bioassay-guided fractionation and the most active fractions were characterized. One bioactive fraction with high cytotoxicity against HT-29 human colon cancer cells (IC50 = 4.0 µg mL-1) was found, and it was characterized as a mixture of p-hydroxybenzoic acid and 4-vinyl-phenol. The I. edulis fruit peel (IC50 = 18.6 µg mL-1) and I. laurina seed (IC50 = 15.2 µg mL-1) extracts had cytotoxic activity against the cell line T98G, and its chemical composition showed a variety of phenolic acids. The chemical composition of this species indicated a wide variety of aromatic acids, flavonoids, tannins, and carotenoids. The high concentration (ranging from 5% to 30%) of these polyphenols in the bioactive extract may be responsible for the antitumor potential. Regarding the proteomic approach, we detected proteins directly related to the elimination of ROS, DNA repair, expression of tumor proteins, and apoptosis.

Taxifolin and Lucidin as Potential E6 Protein Inhibitors: p53 Function Re-Establishment and Apoptosis Induction in Cervical Cancer Cells.

Cervical cancer is the fourth leading cause of death in women worldwide, with 99% of cases associated with a human papillomavirus (HPV) infection. Given that HPV prophylactic vaccines do not exert a therapeutic effect in individuals previously infected, have low coverage of all HPV types, and have poor accessibility in developing countries, it is unlikely that HPV-associated cancers will be eradicated in the coming years. Therefore, there is an emerging need for the development of anti-HPV drugs. Considering HPV E6's oncogenic role, this protein has been proposed as a relevant target for cancer treatment. In the present work, we employed in silico tools to discover potential E6 inhibitors, as well as biochemical and cellular assays to understand the action of selected compounds in HPV-positive cells (Caski and HeLa) vs. HPV-negative (C33A) and non-carcinogenic (NHEK) cell lines. In fact, by molecular docking and molecular dynamics simulations, we found three phenolic compounds able to dock in the E6AP binding pocket of the E6 protein. In particular, lucidin and taxifolin were able to inhibit E6-mediated p53 degradation, selectively reduce the viability, and induce apoptosis in HPV-positive cells. Altogether, our data can be relevant for discovering promising leads for the development of specific anti-HPV drugs.

Vaccines against Infectious Diseases and Cancer.

We live on a planet marked by remarkable health disparities [...].

In Silico Approaches: A Way to Unveil Novel Therapeutic Drugs for Cervical Cancer Management.

Cervical cancer (CC) is the fourth most common pathology in women worldwide and presents a high impact in developing countries due to limited financial resources as well as difficulties in monitoring and access to health services. Human papillomavirus (HPV) is the leading cause of CC, and despite the approval of prophylactic vaccines, there is no effective treatment for patients with pre-existing infections or HPV-induced carcinomas. High-risk (HR) HPV E6 and E7 oncoproteins are considered biomarkers in CC progression. Since the E6 structure was resolved, it has been one of the most studied targets to develop novel and specific therapeutics to treat/manage CC. Therefore, several small molecules (plant-derived or synthetic compounds) have been reported as blockers/inhibitors of E6 oncoprotein action, and computational-aided methods have been of high relevance in their discovery and development. In silico approaches have become a powerful tool for reducing the time and cost of the drug development process. Thus, this review will depict small molecules that are already being explored as HR HPV E6 protein blockers and in silico approaches to the design of novel therapeutics for managing CC. Besides, future perspectives in CC therapy will be briefly discussed.

Antifungal activity of 2'-hydroxychalcone loaded in nanoemulsion against Paracoccidioides spp.

Aim: This study aimed to evaluate the activity of 2'-hydroxychalcone-loaded in nanoemulsion (NLS + 2'chalc), the cytotoxic effect and toxicity against Paracoccidioides brasiliensis and Paracoccidioides lutzii using a zebrafish model. Materials & methods: Preparation and physical-chemical characterization of nanoemulsion (NLS) and NLS + 2'chalc were performed. MIC and minimum fungicide concentration, cytotoxicity and toxicity were also evaluated in the Danio rerio model. Results: NLS + 2'chalc showed fungicidal activity against Paracoccidioides spp. without cytotoxicity in MRC5 and HepG2 lines. It also had high selectivity index values and no toxicity in the zebrafish model based on MIC values. Conclusion: NLS + 2'chalc is a potential new alternative treatment for paracoccidioidomycosis.

Histone Deacetylase Inhibitors as Therapeutic Interventions on Cervical Cancer Induced by Human Papillomavirus.

The role of epigenetic modifications on the carcinogenesis process has received a lot of attention in the last years. Among those, histone acetylation is a process regulated by histone deacetylases (HDAC) and histone acetyltransferases (HAT), and it plays an important role in epigenetic regulation, allowing the control of the gene expression. HDAC inhibitors (HDACi) induce cancer cell cycle arrest, differentiation, and cell death and reduce angiogenesis and other cellular events. Human papillomaviruses (HPVs) are small, non-enveloped double-stranded DNA viruses. They are major human carcinogens, being intricately linked to the development of cancer in 4.5% of the patients diagnosed with cancer worldwide. Long-term infection of high-risk (HR) HPV types, mainly HPV16 and HPV18, is one of the major risk factors responsible for promoting cervical cancer development. In vitro and in vivo assays have demonstrated that HDACi could be a promising therapy to HPV-related cervical cancer. Regardless of some controversial studies, the therapy with HDACi could target several cellular targets which HR-HPV oncoproteins could be able to deregulate. This review article describes the role of HDACi as a possible intervention in cervical cancer treatment induced by HPV, highlighting the main advances reached in the last years and providing insights for further investigations regarding those agents against cervical cancer.

Highlights in Endocytosis of Nanostructured Systems.

The focus of this review is the cellular internalisation mechanism of nanostructured systems (NSs) and their endosomal escape for targeted drug delivery. Endocytosis is a cellular process of internalisation of different molecules and foreign microorganisms. It is currently being studied for drug delivery through nanostructured systems. The most commonly studied routes of cellular uptake are phagocytosis, macro-pinocytosis, clathrinmediated endocytosis, caveolin-mediated endocytosis, and clathrin and caveolinindependent endocytosis. The mechanism utilised by NSs for cellular entry depends on factors such as cell type and its physicochemical properties. Currently, with the development of drugs-loaded onto NSs, it has been possible to increase the therapeutic index against few diseases. The NSs can deliver the active drug at locations that conventional drugs cannot, thereby minimising unwanted side effects. On cellular entry of NSs, there is a possibility of an endosomal escape of the contents into the cytoplasm, a mechanism that can be exploited so that NSs can migrate intra-cellularly and deliver the drug to the target of interest. Designing endolysosomal escape strategy is not an easy task, but it is critical for the optimal pharmacological action on the target tissue. The cellular uptake of drugs is a very important factor in therapy. Although NSs have emerged as effective drug delivery vehicle for treatment of diseases, it is crucial to understand the mechanism of NSs endocytosis.

Synthesis of the Peptide Ac-Wahx-KTTKS and Evaluation of the Ability to Induce In Vitro Collagen Synthesis.

In this work, we report the synthesis of a peptide analogue of the KTTKS, termed Ac- Wahx-KTTKS and evaluate its cytotoxicity and role in biosynthesis of collagen for future application in skin aging. The peptide was obtained with purity higher than 97.5%. In the cytotoxicity assay, we observed non-toxic effects for Ac-WAhx-KTTKS at concentrations below 600 µM for HaCaT and 500 µM for HepG2 cells, respectively. After 24 and 48 h it was possible to observe significant changes in collagen synthesis in the groups treated with various concentrations of the peptide. In conclusion, the Ac-Wahx- KTTKS peptide increased collagen synthesis in fibroblasts by 80% and it is a promising candidate for improving skin aging.

Nanoparticles increase the efficacy of cancer chemopreventive agents in cells exposed to cigarette smoke condensate.

Lung cancer is a leading cause of death in developed countries. Although smoking cessation is a primary strategy for preventing lung cancer, former smokers remain at high risk of cancer. Accordingly, there is a need to increase the efficacy of lung cancer prevention. Poor bioavailability is the main factor limiting the efficacy of chemopreventive agents. The aim of this study was to increase the efficacy of cancer chemopreventive agents by using lipid nanoparticles (NPs) as a carrier. This study evaluated the ability of lipid NPs to modify the pharmacodynamics of chemopreventive agents including N-acetyl-L-cysteine, phenethyl isothiocyanate and resveratrol (RES). The chemopreventive efficacy of these drugs was determined by evaluating their abilities to counteract cytotoxic damage (DNA fragmentation) induced by cigarette smoke condensate (CSC) and to activate protective apoptosis (annexin-V cytofluorimetric staining) in bronchial epithelial cells both in vitro and in ex vivo experiment in mice. NPs decreased the toxicity of RES and increased its ability to counteract CSC cytotoxicity. NPs significantly increased the ability of phenethyl isothiocyanate to attenuate CSC-induced DNA fragmentation at the highest tested dose. In contrast, this potentiating effect was observed at all tested doses of RES, NPs dramatically increasing RES-induced apoptosis in CSC-treated cells. These results provide evidence that NPs are highly effective at increasing the efficacy of lipophilic drugs (RES) but are not effective towards hydrophilic agents (N-acetyl-L-cysteine), which already possess remarkable bioavailability. Intermediate effects were observed for phenethyl isothiocyanate. These findings are relevant to the identification of cancer chemopreventive agents that would benefit from lipid NP delivery.

An Intracellular Arrangement of Histoplasma capsulatum Yeast-Aggregates Generates Nuclear Damage to the Cultured Murine Alveolar Macrophages.

Histoplasma capsulatum is responsible for a human systemic mycosis that primarily affects lung tissue. Macrophages are the major effector cells in humans that respond to the fungus, and the development of respiratory disease depends on the ability of Histoplasma yeast cells to survive and replicate within alveolar macrophages. Therefore, the interaction between macrophages and H. capsulatum is a decisive step in the yeast dissemination into host tissues. Although the role played by components of cell-mediated immunity in the host's defense system and the mechanisms used by the pathogen to evade the host immune response are well understood, knowledge regarding the effects induced by H. capsulatum in host cells at the nuclear level is limited. According to the present findings, H. capsulatum yeast cells display a unique architectural arrangement during the intracellular infection of cultured murine alveolar macrophages, characterized as a formation of aggregates that seem to surround the host cell nucleus, resembling a "crown." This extranuclear organization of yeast-aggregates generates damage on the nucleus of the host cell, producing DNA fragmentation and inducing apoptosis, even though the yeast cells are not located inside the nucleus and do not trigger changes in nuclear proteins. The current study highlights a singular intracellular arrangement of H. capsulatum yeast near to the nucleus of infected murine alveolar macrophages that may contribute to the yeast's persistence under intracellular conditions, since this fungal pathogen may display different strategies to prevent elimination by the host's phagocytic mechanisms.

Assessment of the chemopreventive effect of casearin B, a clerodane diterpene extracted from Casearia sylvestris (Salicaceae).

Studies have shown that Casearia sylvestris compounds protect DNA from damage both in vitro and in vivo. Complementarily, the aim of the present study was to assess the chemopreventive effect of casearin B (CASB) against DNA damage using the Ames test, the comet assay and the DCFDA antioxidant assay. The genotoxicity was assessed by the comet assay in HepG2 cells. CASB was genotoxic at concentrations higher than 0.30 µM when incubated with the FPG (formamidopyrimidine-DNA glycosylase) enzyme. For the antigenotoxicity comet assay, CASB protected the DNA from damage caused by H(2)O(2) in the HepG2 cell line in concentrations above 0.04 µM with post-treatment, and above 0.08 µM with pre-treatment. CASB was not mutagenic (Ames test) in TA 98 and TA 102. In the antimutagenicity assays, the compound was a strong inhibitor against aflatoxin B1 (AFB) in TA 98 (>88.8%), whereas it was moderate (42.7-59.4%) inhibitor against mytomicin C (MMC) in TA 102. Additionally, in the antioxidant assay using DCFDA, CASB reduced reactive oxygen species (ROS) generated by H(2)O(2). In conclusion, CASB was genotoxic to HepG2 cells at high concentrations; was protective of DNA at low concentrations, as shown by the Ames test and comet assay; and was also antioxidant.

Possible implication of Mdm2 as a prognostic marker in invasive laryngeal carcinoma.

Laryngeal squamous cell carcinoma is one of the most common malignant neoplasms of the head and neck. In Brazil, laryngeal tumors represent 2% of all cancers and are associated with approximately 3,000 deaths annually. Human papillomavirus (HPV) has been reported to play an important role in the etiology of laryngeal cancer. The aim of the present study was to evaluate the expression of p53, p27, and Mdm2 in laryngeal carcinomas. Sixty-three larynx biopsies were selected for the study, including 9 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastasis and 27 laryngeal carcinomas with metastasis. Twenty-seven cervical lymph nodes from patients with metastatic lesions were also evaluated. The expression levels of p53, p27, and Mdm2 were assessed by immunohistochemistry using a computer-assisted system. HPV detection and typing were performed using PCR, and the HPV types that were evaluated included HPV 6, 11, 16, 18, 31 and 33. Out of 63 patients, 53 (84.1%) were positive for ß-globin (internal control), and 10 (15.9%) were ß-globin negative and therefore excluded from the evaluation. Thus, 7 (13.2%) out of 53 patients were HPV positive, and 46 (86.8%) out of 53 patients were HPV negative. Statistically significant differences (p < 0.05) in Mdm2 expression levels were observed in the in situ laryngeal carcinoma samples compared with the laryngeal carcinoma samples with metastasis. No statistically significant differences (p > 0.05) in either p53 or p27 expression levels were detected. These findings suggest that Mdm2 may be associated with the invasiveness and aggressiveness of laryngeal carcinomas.

Expression of nonclassical molecule human leukocyte antigen-G in oral lesions.

INTRODUCTION: Human leukocyte antigen (HLA)-G is a nonclassic class I molecule that acts as a modulator of immune responses, and the expression of these molecules in virus-infected cells has been associated with subversion of the immune response. OBJECTIVE: In this study, we performed a cross-sectional study, systematically comparing the expression of the HLA-G in benign, premalignant, and malignant oral lesions and correlating it with the presence of high-risk and low-risk human papillomavirus (HPV) types. SPECIMENS AND METHODS: Oral biopsies were collected from 51 patients and analyzed by immunohistochemistry using anti-HLA-G antibody. Human papillomavirus detection and typing from oral biopsies were obtained by polymerase chain reaction using GP5+/GP6+ and specific primers. RESULTS: The 51 biopsies were stratified into 3 groups according to lesion grade: oral benign lesions (oral hyperplasia and papilloma, n = 16), oral premalignant lesions (oral leukoplakia with dysplasia and lichen planus, n = 17), and malignant lesions (oral squamous cell carcinoma, n = 18). Human leukocyte antigen-G overexpression was mainly observed in benign and premalignant oral lesions but was not related to HPV infection (P > .05). On the other hand, HPV DNA was detected in 24 (47%) oral lesions, mainly in benign and premalignant lesions, with the most frequent type detected being high-risk HPV type. CONCLUSION: The HLA-G molecule was expressed in a significant number of benign oral lesions and was not correlated with HPV infection or oral cancer.

Expression of the nonclassical HLA-G and HLA-E molecules in laryngeal lesions as biomarkers of tumor invasiveness.

INTRODUCTION: HLA-G and HLA-E are two nonclassical class I molecules, which have been well recognized as modulators of innate and adaptive immune responses, and the expression of these molecules in virus infected cells has been associated with subversion of the immune response. OBJECTIVE: In this study we performed a cross-sectional study, systematically comparing the expression of HLA-G and HLA-E in benign, pre-malignant and malignant laryngeal lesions, correlating with demographic and clinical variables and with the presence of high-risk and low-risk HPV types. MATERIALS AND METHODS: Laryngeal lesions were collected from 109 patients and stratified into 27 laryngeal papillomas, 17 dysplasias, 10 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastases, 28 laryngeal carcinomas with metastasis along with their respective draining cervical lymph nodes, and 10 normal larynx specimens. The expression of HLA-G and HLA-E molecules was determined by immunohistochemistry. HPV DNA detection and typing was performed using generic and specific primers. RESULTS: HLA nonclassical molecules showed a distinct distribution pattern, according to the larynx lesion grade. HLA-G expression increased in benign and premalignant lesions, and gradually decreased in invasive carcinomas and in respective draining cervical lymph nodes. Conversely, HLA-E expression increased as far as lesion grade increased, including increased molecule expression in the draining lymph nodes of malignant lesions. Only 17 (15.6%) patients were HPV DNA positive. CONCLUSIONS: Overexpression of HLA-E and underexpression of HLA-G appear to be good markers for malignant larynx lesion.

Suppression of TNF-α induced NFκB activity by gallic acid and its semi-synthetic esters: possible role in cancer chemoprevention.

Gallic acid (3,4,5-trihydroxybenzoic acid), found in many plants either in free-form or part of tannins, is known to possess anti-microbial, antioxidant and cytotoxic properties. NFκB regulates the expression of several genes involved in carcinogenesis. These include anti-apoptotic, cytokines and cell cycle-regulatory genes. It is well established that the transcriptional factor NFκB is deregulated in many forms of cancer. Thus, agents that can suppress NFκB activation have the potential of suppressing carcinogenesis. In the present investigation, gallic acid was isolated from Alchornea glandulosa (Euphorbiaceae) and eight esters were synthesised. These compounds were evaluated against TNF-α-induced NFκB activation with stably transfected 293/NFκB-Luc human embryonic kidney cells. Gallates with IC(50) values in a range of 10-56 µM mediated inhibitory activity higher than gallic acid (IC(50) 76.0 ± 4.9 µM). In addition to inhibiting NFκB activation, gallic acid mediated a modest cytotoxic effect, and some of the gallates affected cell viability at the tested concentrations. Based on these results, suppression of NFκB activation by gallate esters could play a chemopreventive role in carcinogenesis.

BUBR1 expression in benign oral lesions and squamous cell carcinomas: correlation with human papillomavirus.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is for 14,160 new cases in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BUBR1, an important protein in the mitotic spindle assembly checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BUBR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BUBR1 in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis and in 16 non-malignant oral lesions. PCR was performed in order to detect HPV DNA. Significantly higher BUBR1 expression associated with shorter survival (p=0.0479) was observed in malignant lesions. There was also a significant correlation (r=1.000) with BUBR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV16 and HVP18 were present in 13 and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with a high BUBR1 expression and the absence of viral DNA had no influence on BUBR1 expression. These findings suggest that HPV could be associated with overexpression of BUBR1 in OSCC, but not in benign oral lesions.

Low expression of human histocompatibility soluble leukocyte antigen-G (HLA-G5) in invasive cervical cancer with and without metastasis, associated with papilloma virus (HPV).

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class Ib molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICC(W))] and absence [ICC without metastasis (ICC(WT))] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICC(WT) (n=52 patients) and ICC(W) (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies.

Ethanolic extract of Casearia sylvestris and its clerodane diterpen (caseargrewiin F) protect against DNA damage at low concentrations and cause DNA damage at high concentrations in mice's blood cells.

Casearia sylvestris is used in Brazil as a popular medicine to treat ulcer, inflammation and tumour. Caseargrewiin F is a clerodane diterpene isolated from the ethanolic leaf extract of C.sylvestris. The aim of the study was to assess the capacity of the ethanolic extract of C.sylvestris leaves and caseargrewiin F to protect DNA and verify if both the compounds cause some DNA damage, using the micronucleus (MN) test and comet assay in mice. Balb-C mice were treated with the extract [3.13, 6.25, 12.5, 25, 50 and 75 mg/kg body weight (b.w.)] and caseargrewiin F (0.16, 0.32, 0.63, 1.3, 2.5 and 3.8 mg/kg b.w.) for 14 days. On day 15, DNA damage was induced by intra-peritoneal (i.p.) injection of cyclophosphamide (CP) (i.p.) at 50 mg/kg b.w. after the MN test and comet assay were performed. A protective effect of ethanolic extract was observed in MN test (6.25 and 12.5 mg/kg b.w.) and the comet assay (3.13 and 6.25, 12.5 and 25 mg/kg b.w.). Caseargrewiin F showed protective effect at 0.63, 1.3 and 2.5 mg/kg b.w. only in comet assay. We also tested the ability of compounds of C.sylvestris to induce MN and to increase the comet assay tail moment. The experimental design was similar to the DNA protection assay except that in test groups we omitted the CP challenge. We observed increased damage at 50 and 75 mg/kg b.w. of ethanolic extract of C.sylvestris and caseargrewiin F at 3.18 mg/kg b.w. in both the MN test and comet assay. We conclude that ethanolic extract of C. sylvestris and caseargrewiin F can protect cells against DNA damage induced by CP at low concentrations, but at high concentrations these compounds also induce DNA damage.