TiO2 NPs Induce DNA Damage in Lymphocytes from Healthy Individuals and Patients with Respiratory Diseases-An Ex Vivo/In Vitro Study.

Little is known of the effects of nanoparticles in human systems, let alone in diseased individuals and nanotechnology has preceded nanotoxicology. Therefore, the effects of titanium dioxide (TiO2) nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases [lung cancer, chronic obstructive pulmonary disease (COPD) and asthma] were compared with those in healthy Individuals, to determine differences in sensitivity to nanochemical insult. The Comet assay was performed according to recommended guidelines. The micronucleus assay and ras oncoprotein detection were conducted according to published standard methods. The results showed statistically significant concentration-dependent genotoxic effects of TiO2 NPs in both respiratory patient and control groups in the Comet assay. The TiO2 NPs caused DNA damage in a concentration dependent manner in both groups (respiratory and healthy controls) with the exception of the lowest TiO2 concentration (10 µg/ml) which did not induce significant damage in healthy controls (n.s). When OTM data were used to compare the whole patient group and the control group, the patient group had more DNA damage (p > 0.001) with the exception of 10 µg/ml of TiO2 that caused less significant damage to patient lymphocytes (p < 0.05). Similarly, there was an increase in the pattern of cytogenetic damage measured in the MN assay without statistical significance except when compared to the negative control of healthy individuals. Furthermore, when modulation of ras p21 expression was investigated, regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels. Results were achieved in the absence of cytotoxicity.

Monitoring characteristics and genotoxic effects of engineered nanoparticle-protein corona.

Engineered nanoparticles (ENPs) possess different physical and chemical properties compared to their bulk counterparts. These unique properties have found application in various products in the area of therapeutics, consumer goods, environmental remediation, optical and electronic fields. This has also increased the likelihood of their release into the environment thereby affecting human health and ecosystem. ENPs, when in contact with the biological system have various physical and chemical interactions with cellular macromolecules including proteins. These interactions lead to the formation of protein corona around the ENPs. Consequently, living systems interact with the protein-coated ENP rather than with a bare ENP. This ENP-protein interaction influences uptake, accumulation, distribution and clearance and thereby affecting the cytotoxic and genotoxic responses. Although there are few studies which discussed the fate of ENPs, there is a need for extensive research in the field of ENPs, to understand the interaction of ENPs with biological systems for their safe and productive application.

Laboratory Scale Microbial Food Chain To Study Bioaccumulation, Biomagnification, and Ecotoxicity of Cadmium Telluride Quantum Dots.

The increasing applications of engineered nanomaterials (ENMs) in consumer products warrant a careful evaluation of their trophic transfer and consequent ecological impact. In the present study, a laboratory scale aquatic microbial food chain was established using bacteria (Escherichia coli (E. coli)) as a prey and ciliated protozoan (Paramecium caudatum) as a predator organism to determine the impact of cadmium telluride quantum dots (CdTe QDs). We observed that 29% of bacterivory potential of paramecium was lost, including an ∼12 h delay in doubling time on exposure to 25 mg/L CdTe QD (∼4 nm) as compared to control. The fluorescence based stoichiometric analysis revealed that 65% of the QDs bioaccumulated when paramecia were exposed to 25 mg/L QDs at 24 h. There was a significant (p < 0.05) increase in cellular cadmium (Cd) concentration at 24 h (306 ± 192 mg/L) as compared to 1 h (152 ± 50 mg/L). Moreover, the accumulation of Cd in E. coli (147 ± 25 mg/L) at 1 h of exposure to 25 mg/L QDs transferred 1.4 times higher Cd (207 ± 24 mg/L; biomagnification factor = 1.4) to its predator, paramecium.

Cell cycle dependent cellular uptake of zinc oxide nanoparticles in human epidermal cells.

Metal oxide nanoparticles (NPs), including zinc oxide (ZnO) NPs have shown success for use as vehicles for drug delivery and targeting gene delivery in many diseases like cancer. Current anticancer chemotherapeutics fail to effectively differentiate between cancerous and normal cells. There is an urgent need to develop novel drug delivery system that can better target cancer cells while sparing normal cells and tissues. Particularly, ZnO NPs exhibit a high degree of cancer cell selectivity and induce cell death, oxidative stress, interference with the cell cycle progression and genotoxicity in cancerous cells. In this scenario, effective cellular uptake of NP seems to be crucial, which is shown to be affected by cell cycle progression. In the present study, the cytotoxic potential of ZnO NPs and the effect of different cell cycle phases on the uptake of ZnO NPs were examined in A431 cells. It is shown that the ZnO NPs led to cell death and reactive oxygen species generation and were able to induce cell cycle arrest in S and G2/M phase with the higher uptake in G2/M phase compared with other phases.

Zinc oxide nanoparticles affect the expression of p53, Ras p21 and JNKs: an ex vivo/in vitro exposure study in respiratory disease patients.

Zinc oxide (ZnO) nanoparticles are the mostly used engineered metal oxide nanoparticles in consumer products. This has increased the likelihood of human exposure to this engineered nanoparticle (ENPs) through different routes. At present, the majority of the studies concerning ZnO ENPs toxicity have been conducted using in vitro and in vivo systems. In this study, for the first time we assessed the effect of ZnO ENPs on the major cellular pathways in the lymphocytes of healthy individuals as well as in susceptible patients suffering from lung cancer, chronic obstructive pulmonary disease (COPD) and asthma. Using the differential expression analysis, we observed a significant (P < 0.05) dose-dependent (10, 20 and 40 µg/ml for 6h) increase in the expression of tumour suppressor protein p53 (40, 60 and 110%); Ras p21 (30, 52 and 80%); c-Jun N-terminal kinases; JNKs) (28, 47 and 78%) in lung cancer patient samples treated with ZnO ENPs compared to healthy controls. A similar trend was also seen in COPD patient samples where a significant (P < 0.05) dose-dependent increase in the expression of tumour suppressor protein p53 (26, 45 and 84%), Ras p21 (21, 40 and 77%), JNKs (17, 32 and 69%) was observed after 6h of ZnO ENPs treatment at the aforesaid concentrations. However, the increase in the expression profile of tested protein was not significant in the asthma patients as compared to controls. Our results reiterate the concern about the safety of ZnO ENPs in consumer products and suggest the need for a complete risk assessment of any new ENPs before its use.

Chromium oxide nanoparticle-induced genotoxicity and p53-dependent apoptosis in human lung alveolar cells.

Chromium oxide (Cr2 O3 ) nanoparticles (NPs) are being increasingly used as a catalyst for aromatic compound manufacture, abrading agents and as pigments (e.g., Viridian). Owing to increased applications, it is important to study the biological effects of Cr2 O3 NPs on human health. The lung is one of the main exposure routes to nanomaterials; therefore, the present study was designed to determine the genotoxic and apoptotic effect of Cr2 O3 NPs in human lung epithelial cells (A549). The study also elucidated the molecular mechanism of its toxicity. Cr2 O3 NPs led to DNA damage, which was deduced by comet assay and cytokinesis block micronucleus assay. The damage could be mediated by the increased levels of reactive oxygen species. Further, the oxygen species led to a decrease in mitochondrial membrane potential and an increase in the ratio of BAX/Bcl-2 leading to mitochondria-mediated apoptosis induced by Cr2 O3 NPs, which ultimately leads to cell death. Hence, there is a need of regulations to be imposed in NP usage. The study provided insight into the caspase-dependent mechanistic pathway of apoptosis.

The comet assay as a tool for human biomonitoring studies: the ComNet project.

The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, comet assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available comet assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the comet assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view.

Hydration patterns of graphene-based nanomaterials (GBNMs) play a major role in the stability of a helical protein: a molecular dynamics simulation study.

Graphene-based nanomaterials (GBNMs) [graphene oxide (GO), reduced graphene oxide (rGO), and graphene] have been recognized as potential candidates for various biomedical applications ranging from biosensing platform to cellular delivery of proteins and peptides. However, GBNMs induced conformational changes in proteins are the major concerns in realizing their full potential in aforementioned applications. Despite several studies, the effect of GBNMs on the conformation of proteins is still not well understood. Therefore, an attempt was made to investigate the effect of GBNMs on the adsorption and conformation of positively charged cytoplasmic protein using molecular dynamics (MD) simulations. Our study showed that the adsorption of protein on GO was highly selective and mediated through electrostatic interactions (hydrogen bond/salt bridge interactions), whereas the van der Waals and π-π stacking interactions were the major driving forces for the adsorption of protein on rGO and graphene. The secondary structure analysis showed the conformational stability of the protein on GO may be attributed to the extensive hydration of GO surface and the absence of tyrosine residues in π-π stacking with π regions of GO. The GO surface acts as a hydrogen bond acceptor similar to the protein's natural receptor present in a physiological environment. This computational study has also explored the artificial protein receptor like potential of GO.

Genotoxic and carcinogenic potential of engineered nanoparticles: an update.

Nanoscience and nanotechnology have seen an exponential growth over the past decade largely due to the unique properties of engineered nanoparticles (ENPs), advances in ENP synthesis, and imaging or analysis tools. The unique properties such as high surface area to volume ratio, abundant reactive sites on the surface, large fraction of atoms located on the exterior face have made these novel materials the most sought after for consumer and industrial applications. This significant increase in the ENP containing consumer products has also enhanced the chances of human and environmental exposure. Humans get exposed to ENPs at various steps of its synthesis (laboratory), manufacture (industry), use (consumer products, devices, medicines, etc.) and through the environment (contaminated water, aerosolized particles, and disposal). Such exposures to ENPs are known to induce genotoxicity, cytotoxicity, and carcinogenicity in biological system. This is attributed to several factors, such as direct interaction of ENPs with the genetic material, indirect damage due to reactive oxygen species generation, release of toxic ions from soluble ENPs, interaction with cytoplasmic/nuclear proteins, binding with mitotic spindle or its components, increased oxidative stress, disturbance of cell cycle checkpoint functions, inhibition of antioxidant defense, and many others. The present review describes an overview of in vitro and in vivo genotoxicity studies with ENPs, advantages and potential problems associated with the methods used in genotoxicity assessment, and the need for appropriate method and approach for risk assessment of ENPs.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2).

The wide scale use of Zinc oxide (ZnO) nanoparticles in the world consumer market makes human beings more prone to the exposure to ZnO nanoparticles and its adverse effects. The liver, which is the primary organ of metabolism, might act as a major target organ for ZnO nanoparticles after they gain entry into the body through any of the possible routes. Therefore, the aim of the present study was to assess the apoptotic and genotoxic potential of ZnO nanoparticles in human liver cells (HepG2) and the underlying molecular mechanism of its cellular toxicity. The role of dissolution in the toxicity of ZnO nanoparticles was also investigated. Our results demonstrate that HepG2 cells exposed to 14-20 µg/ml ZnO nanoparticles for 12 h showed a decrease in cell viability and the mode of cell death induced by ZnO nanoparticles was apoptosis. They also induced DNA damage which was mediated by oxidative stress as evidenced by an increase in Fpg sensitive sites. Reactive oxygen species triggered a decrease in mitochondria membrane potential and an increase in the ratio of Bax/Bcl2 leading to mitochondria mediated pathway involved in apoptosis. In addition, ZnO nanoparticles activated JNK, p38 and induced p53(Ser15) phosphorylation. However, apoptosis was found to be independent of JNK and p38 pathways. This study investigating the effects of ZnO nanoparticles in human liver cells has provided valuable insights into the mechanism of toxicity induced by ZnO nanoparticles.

Induction of oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure to zinc oxide nanoparticles.

Zinc oxide (ZnO) nanoparticles are finding applications in a wide range of products including cosmetics, food packaging, imaging, etc. This increases the likelihood of human exposure to these nanoparticles through dermal, inhalation and oral routes. Presently, the majority of the studies concerning ZnO nanoparticle toxicity have been conducted using in vitro systems which lack the complex cell-cell, cell-matrix interactions and hormonal effects found in the in vivo scenario. The present in vivo study in mice was aimed at investigating the oral toxicity of ZnO nanoparticles. Our results showed a significant accumulation of nanoparticles in the liver leading to cellular injury after sub-acute oral exposure of ZnO nanoparticles (300 mg/kg) for 14 consecutive days. This was evident by the elevated alanine aminotransferase (ALT) and alkaline phosphatase (ALP) serum levels and pathological lesions in the liver. ZnO nanoparticles were also found to induce oxidative stress indicated by an increase in lipid peroxidation. The DNA damage in the liver and kidney cells of mice was evaluated by the Fpg-modified Comet assay which revealed a significant (p<0.05) increase in the Fpg-specific DNA lesions in liver indicating oxidative stress as the cause of DNA damage. The TUNEL assay revealed an induction of apoptosis in the liver of mice exposed to ZnO nanoparticles compared to the control. Our results conclusively demonstrate that sub-acute oral exposure to ZnO nanoparticles in mice leads to an accumulation of nanoparticles in the liver causing oxidative stress mediated DNA damage and apoptosis. These results also suggest the need for a complete risk assessment of any new engineered nanoparticle before its arrival into the consumer market.

DNA and oxidative damage induced in somatic organs and tissues of mouse by municipal sludge leachate.

Pollution by waste landfill leachate has prompted a number of studies on the toxic and potential health effects. This study assessed the genotoxicity of a municipal sludge leachate (MSL) in the somatic tissues (blood and bone marrow) and organs (liver, kidney, and spleen) of mice using the alkaline Comet assay. The possible cause of DNA damage via the study of antioxidant system (lipid peroxidation [LPO]; catalase [CAT]; reduced glutathione [GSH]; and superoxide dismutase [SOD]) responses in mouse liver was also investigated. Different concentrations (2.5%, 5%, 10%, and 15%) of the leachate were administered intraperitoneally for 5 consecutive days to male Swiss albino mice (4 mice/group). A significant (p < 0.05) increase in DNA damage in organs and tissues of treated mice compared to the negative control was observed as evident from the Comet assay parameters: olive tail moment (OTM, arbitrary units) and tail DNA (%). Bone marrow showed maximum DNA damage followed by liver > spleen > kidney > blood as evident by the OTM. A significant increase (p < 0.05) in the level of antioxidant enzymes (CAT and SOD) and LPO with a concurrent decrease in GSH in the liver of treated mice was also observed. Our finding demonstrates that the MSL induces DNA damage in the somatic tissues and organs of mouse as well as induces oxidative stress in the liver. These tissues and organs may be the potential targets in animal and human populations exposed to MSL. This is of relevance to public health; as such exposure could lead to adverse health effects via systemic genotoxicity.

Ultrastructural Changes in Autopsy Tissues of COVID-19 Patients.

INTRODUCTION: The COVID-19 pandemic resulted in substantial morbidity and mortality across the world. The prognosis was found to be poor in patients with co-morbidities such as diabetes, hypertension, interstitial lung disease, etc. Although biochemical studies were done in patient samples, no study has been reported from the Indian subcontinent about ultrastructural changes in the vital organs of COVID-19 patients. The present study was, therefore, conducted to understand the ultrastructural changes in the lung, liver, and brain of the deceased patients. METHODS: The present study was conducted on samples obtained from reverse transcription-polymerase chain reaction (RT-PCR)-positive patients who were admitted to a tertiary care hospital in Western India. Core needle biopsies were done in eight fatal cases of COVID-19. The samples were taken from the lungs, liver, and brain and subjected to light microscopy, immunohistochemistry (IHC), and transmission electron microscopy (TEM). Clinical details and biochemical findings were also collected.  Results: The study participants included seven males and one female. The presenting complaints included fever, breathlessness, and cough. Light microscopy revealed diffuse alveolar damage in the lungs. Further, a positive expression of SARS-CoV-2 nucleocapsid protein was observed in the pulmonary parenchyma of five patients. Also, the TEM microphotograph showed viral particles of size up to 80nm localized in alveolar epithelial cells. However, no viral particles were found in liver or brain samples. In the liver, macrovesicular steatosis and centrizonal congestion with loss of hepatocytes were observed in light microscopy. CONCLUSION:  This is the first study in the Indian population showing the in-situ presence of viral particles in core biopsies from fatal cases of COVID-19. As evident from the results, histology and ultrastructural changes in the lung correlated with the presence of viral particles. The study revealed a positive correlation between the damage in the lungs and the presence of viral particles.

A flow cytometric method to assess nanoparticle uptake in bacteria.

Toxicity of engineered nanomaterials (ENMs), such as metal oxides, has been of concern among environmental and health scientists. For ecotoxicity studies of ENMs, it is important to assess nanoparticle uptake and correlate it with the cellular response. However, due to nonavailability of adequate methods for assessing cellular uptake of ENMs, there is a lack of information in this important area. In the present study, a method has been developed using flow cytometry, which allows for rapid detection of ENM internalization in live bacteria under different experimental conditions for several generations. Our data demonstrate significant internalization of Zinc oxide (ZnO) and Titanium (IV) oxide (TiO(2) ) nanoparticles (NPs) in Escherichia coli in a dose-dependent manner. ZnO NPs treatment exhibited a significant increase in the intensity of side scatter (SSC) with liver-S9 fraction (76, 94, and 181% increase) rather than without S9 (10.5, 24.5, and 125.9% increase) at 10, 40, and 80 µg/ml concentrations, respectively. This was due to the protein coating of NPs by the S9 fraction. A similar response was also observed on exposure to TiO(2) NPs (139 and 203% with S9 and 128 and 198% without S9). In a multigeneration study, this new method was able to detect the presence of ENMs in E. coli up to four generations. Our data demonstrate that this method can be used for assessing the uptake of ENMs in bacteria and provides a handle to toxicologists for ecotoxicity studies of economically important ENMs to ensure safer products in the market.

Zinc oxide nanoparticle induced genotoxicity in primary human epidermal keratinocytes.

Zinc oxide (ZnO) nanoparticles are widely used in cosmetics and sunscreens. Human epidermal keratinocytes may serve as the first portal of entry for these nanoparticles either directly through topically applied cosmetics or indirectly through any breaches in the skin integrity. Therefore, the objective of the present study was to assess the biological interactions of ZnO nanoparticles in primary human epidermal keratinocytes (HEK) as they are the most abundant cell type in the human epidermis. Cellular uptake of nanoparticles was investigated by scanning electron microscopy using back scattered electrons imaging as well as transmission electron microscopy. The electron microscopy revealed the internalization of ZnO nanoparticles in primary HEK after 6 h exposure at 14 microg/ml concentration. ZnO nanoparticles exhibited a time (6-24 h) as well as concentration (8-20 microg/ml) dependent inhibition of mitochondrial activity as evident by the MTT assay. A significant (p < 0.05) induction in DNA damage was observed in cells exposed to ZnO nanoparticles for 6 h at 8 and 14 microg/ml concentrations compared to control as evident in the Comet assay. This is the first study providing information on biological interactions of ZnO nanoparticles with primary human epidermal keratinocytes. Our findings demonstrate that ZnO nanoparticles are internalized by the human epidermal keratinocytes and elicit a cytotoxic and genotoxic response. Therefore, caution should be taken while using consumer products containing nanoparticles as any perturbation in the skin barrier could expose the underlying cells to nanoparticles.

Expression profiling of toxicity pathway genes by real-time PCR array in cypermethrin-exposed mouse brain.

Cypermethrin, a type II pyrethroid, has been shown to exert genotoxic effects in the central nervous system of non-target species such as mouse and Drosophila. To unravel the gene expression of toxicity-related pathways in cypermethrin-exposed Swiss albino mouse brain, transcriptional profiling was carried out through pathway-focused real-time PCR arrays (DNA damage signaling, oxidative stress/antioxidants, and stress/toxicity pathways). The real-time PCR array data revealed a significant (p < 0.05) modulation in transcript levels of 61 genes involved in DNA replication and repair, apoptosis, cell cycle, oxidative stress, and toxicity pathways. Cypermethrin also produced oxidative stress in brain, as was evident by a significant (p < 0.05) elevation (66%) in lipid peroxidation and reduction of glutathione (GSH) content (10.6%) as well as catalase activity (56.7%). The results demonstrate that cypermethrin alters the expression of stress- and toxicity-related genes as well as induces oxidative stress which may lead to DNA damage. These observations also point to complex metabolic networks involved in genotoxic manifestations by cypermethrin.

Toxicity assessment of nanomaterials: methods and challenges.

The increasing use of nanomaterials in consumer and industrial products has aroused global concern regarding their fate in biological systems, resulting in a demand for parallel risk assessment. A number of studies on the effects of nanoparticles in in vitro and in vivo systems have been published. However, there is still a need for further studies that conclusively establish their safety/toxicity, due to the many experimental challenges and issues encountered when assessing the toxicity of nanomaterials. Most of the methods used for toxicity assessment were designed and standardized with chemical toxicology in mind. However, nanoparticles display several unique physicochemical properties that can interfere with or pose challenges to classical toxicity assays. Recently, some new methods and modified versions of pre-existing methods have been developed for assessing the toxicity of nanomaterials. This review is an attempt to highlight some important methods employed in nanomaterial toxicology and to provide a critical analysis of the major issues/challenges faced in this emerging field.

Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p(53) and p(21/WAF1) proteins in skin of mice.

Patulin (PAT), a mycotoxin found in apples, grapes, oranges, pear and peaches, is a potent genotoxic compound. WHO has highlighted the need for the study of cutaneous toxicity of PAT as manual labour is employed during pre and post harvest stages, thereby causing direct exposure to skin. In the present study cutaneous toxicity of PAT was evaluated following topical application to Swiss Albino mice. Dermal exposure of PAT, to mice for 4 h resulted in a dose (40-160 mug/animal) and time (up to 6 h) dependent enhancement of ornithine decarboxylase (ODC), a marker enzyme of cell proliferation. The ODC activity was found to be normal after 12 and 24 h treatment of patulin. Topical application of PAT (160 mug/100 mul acetone) for 24-72 h caused (a) DNA damage in skin cells showing significant increase (34-63%) in olive tail moment, a parameter of Comet assay (b) significant G 1 and S-phase arrest along with induction of apoptosis (2.8-10 folds) as shown by annexin V and PI staining assay through flow cytometer. Moreover PAT leads to over expression of p(21/WAF1) (3.6-3.9 fold), pro apoptotic protein Bax (1.3-2.6) and tumor suppressor wild type p(53) (2.8-3.9 fold) protein. It was also shown that PAT induced apoptosis was mediated through mitochondrial intrinsic pathway as revealed through the release of cytochrome C protein in cytosol leading to enhancement of caspase-3 activity in skin cells of mice. These results suggest that PAT has a potential to induce DNA damage leading to p(53) mediated cell cycle arrest along with intrinsic pathway mediated apoptosis that may also be correlated with enhanced polyamine production as evident by induction of ODC activity, which may have dermal toxicological implications.