Toxicology Evaluation of Drugs Administered via Uncommon Routes: Intranasal, Intraocular, Intrathecal/Intraspinal, and Intra-Articular.

As the need for nasal, ocular, spinal, and articular therapeutic compounds increases, toxicology assessments of drugs administered via these routes play an important role in human safety. This symposium outlined the local and systemic evaluation to support safety during the development of these drugs in nonclinical models with some case studies. Discussions included selection of appropriate species for the intended route; conducting nonclinical studies that closely mimic the intended use with adequate duration; functional assessment, if deemed necessary; evaluation of local tissues with special histological staining procedure; and evaluations of safety margins based on local and systemic toxicity.

Effects of epigallocatechin gallate, L-ascorbic acid, alpha-tocopherol, and dihydrolipoic acid on the formation of deoxyguanosine adducts derived from lipid peroxidation.

Oxidation of polyunsaturated fatty acids (PUFAs) releases alpha,beta-unsaturated aldehydes that modify deoxyguanosine (dG) to form cyclic 1,N(2)-propanodeoxyguanosine adducts. One of the major adducts detected in vivo is acrolein (Acr)-derived 1,N(2)-propanodeoxyguanosine (Acr-dG). We used a chemical model system to examine the effects of 4 antioxidants known to inhibit fatty acid oxidation on the formation of Acr-dG and 8-oxodeoxyguaonsine (8-oxodG) from the PUFA docosahexaenoic acid (DHA) under oxidative conditions. We found that epigallocatechin gallate (EGCG) and dihydrolipoic acid (DHLA) inhibit both Acr-dG and 8-oxodG formation. In contrast, ascorbic acid and alpha-tocopherol actually increase Acr-dG at high concentrations and do not show a concentration-dependant inhibition of 8-oxodG. We also studied their effects on blocking Acr-dG formation directly from Acr. EGCG and DHLA can both effectively block Acr-dG formation, but ascorbic acid and alpha-tocopherol show weak or little effect. These results highlight the complexity of antioxidant mechanisms and also reveal that EGCG and DHLA are effective at suppressing lipid peroxidation-induced Acr-dG and 8-oxodG formation as well as blocking the reaction of dG with Acr.

Acrolein-derived DNA adduct formation in human colon cancer cells: its role in apoptosis induction by docosahexaenoic acid.

The apoptotic effects of docosahexaenoic acid (DHA) and other omega-3 polyunsaturated fatty acids (PUFAs) have been documented in cell and animal studies. The molecular mechanism by which DHA induces apoptosis is unclear. Although there is no direct evidence, some studies have suggested that DNA damage generated through lipid peroxidation may be involved. Our previous studies showed that DHA, because it has a high degree of unsaturation, can give rise to the acrolein-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) as a major class of DNA adducts via lipid oxidation. As a first step to investigate the possible role of oxidative DNA damage in apoptosis induced by DHA, we examined the relationships between oxidative DNA damage and apoptosis caused by DHA in human colon cancer HT-29 cells. Apoptosis and oxidative DNA damage, including Acr-dG and 8-oxo-deoxyguanosine (8-oxo-dG) formation, in cells treated with DHA and omega-6 PUFAs, including arachidonic acid (AA) and linoleic acid (LA), were measured. DHA induced apoptosis in a dose- and time-dependent manner with a concentration range from 0 to 300 microM as indicated by increased caspase-3 activity and PARP cleavage. In contrast, AA and LA had little or no effect at these concentrations. The Acr-dG levels were increased in HT-29 cells treated with DHA at 240 and 300 microM, and the increases were correlated with the induction of apoptosis at these concentrations, while no significant changes were observed for 8-oxo-dG. Because proteins may compete with DNA to react with acrolein, we then examined the effects of BSA on DHA-induced apoptosis and oxidative DNA damage. The addition of BSA to HT-29 cell culture media significantly decreases Acr-dG levels with a concomitant decrease in the apoptosis induced by DHA. The reduced Acr-dG formation is attributed to the reaction of BSA with acrolein as indicated by increased levels of total protein carbonyls. Similar correlations between Acr-dG formation and apoptosis were observed in HT-29 cells directly incubated with 0-200 microM acrolein. Additionally, DHA treatment increased the level of DNA strand breaks and caused cell cycle arrested at G1 phase. Taken together, these results demonstrate the parallel relationships between Acr-dG level and apoptosis in HT-29 cells, suggesting that the formation of Acr-dG in cellular DNA may contribute to apoptosis induced by DHA.

Detection of the acrolein-derived cyclic DNA adduct by a quantitative 32P-postlabeling/solid-phase extraction/HPLC method: blocking its artifact formation with glutathione.

Acrolein (Acr), a hazardous air pollutant, reacts readily with deoxyguanosine (dG) in DNA to produce cyclic 1, N2-propanodeoxyguanosine adducts (Acr-dG). Studies demonstrate that these adducts are detected in vivo and may play a role in mutagenesis and carcinogenesis. In the study described here, a quantitative 32P-postlabeling/solid-phase extraction/HPLC method was developed by optimizing the solid-phase extraction and the 32P-postlabeling conditions for analysis of Acr-dG in DNA samples with a detection limit of 0.1 fmol. It was found that Acr-dG can form as an artifact during the assay. Evidence obtained from mass spectrometry indicates that the Acr in water used in the assay is a likely source of artifact formation of Acr-dG. The formation of Acr-dG as an artifact can be effectively blocked by adding glutathione (GSH) to the DNA sample to be analyzed. In addition, Acr-dG was detected as a contaminant in the commercial dG and dT 3'-monophosphate samples. Finally, this method was used to detect Acr-dG in calf thymus and human colon HT29 cell DNA with an excellent linear quantitative relationship.

P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5.

The death-inducing cytokine TRAIL is a promising agent for anticancer therapy since it preferentially kills cancer versus normal cells; however, some cancer cells are TRAIL-resistant. We initially explored whether overexpression of the MDR1 gene product P-glycoprotein (P-gp), which causes multidrug resistance (MDR) in cancer cells, also contributes to TRAIL-resistance. Surprisingly, our results revealed that P-gp-overexpression enhances TRAIL-induced apoptosis not only in neoplastic cells transfected with the MDR1 gene but also in MDR variants selected with cytotoxic anticancer agents. Mechanistic analysis of TRAIL-induced apoptosis in the MDR1-transfected MCF-7 breast cancer cell line BC-19 revealed that TRAIL-triggered significantly more apoptosis in these cells compared with parental MCF-7 cells by binding to the TRAIL receptor DR5. DR5 but not DR4 engagement by TRAIL attenuated cellular ATP levels by robustly stimulating P-gp ATPase activity, and thus triggered P-gp-dependent apoptosis by depletion of the cellular ATP pool. In addition to hyperactive P-gp-mediated ATP hydrolysis, TRAIL-induced, P-gp-potentiated apoptosis was associated with activation of caspases-6, -7, -8, and -9; Bid cleavage; and mitochondrial depolarization. P-gp interacted with the TRAIL receptors DR4, DR5, and DcR1 in plasma membranes and enhanced TRAIL binding to DR5. Interestingly, the decreased level of the decoy TRAIL receptor, DcR1, in BC-19 cells further sensitized these cells to TRAIL. Therefore, both extrinsic and intrinsic apoptosis pathways are involved in this process. These findings for the first time reveal that TRAIL treatment preferentially causes apoptosis in P-gp-overexpressing MDR cells, and suggests significant clinical implications for the use of TRAIL in treating neoplasms that have failed chemotherapy.

Taxol induces caspase-10-dependent apoptosis.

Taxol (paclitaxel) is known to inhibit cell growth and trigger significant apoptosis in various cancer cells. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. In this study we investigated death receptors, FAS-associated death domain protein (FADD), the activation of caspases-10 and -8 as well as the downstream caspases, and reactive oxygen species (ROS) in taxol-induced apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line. Pretreating the cells with neutralizing antibodies to Fas, tumor necrosis factor (TNF)-alpha receptor 1, or TNF-related apoptosis-inducing ligand receptors (DR4 and DR5) did not affect taxol-induced apoptosis, but transfection of the cells with a dominant negative FADD plasmid resulted in inhibition of taxol-induced apoptosis, revealing that taxol induces apoptosis independently of these death receptors but dependently on FADD. Furthermore, the drug induced activation of caspases-10, -8, -6, and -3, cleaved Bcl-2, Bid, poly(ADP-ribose) polymerase, and lamin B, and down-regulated cellular levels of FLICE-like inhibitory protein (FLIP) and X-chromosome-linked inhibitor of apoptosis protein (XIAP). However, despite the release of cytochrome c from the mitochondria in taxol-treated cells, caspase-9 was not activated. Inhibitors of caspases-8, -6, or -3 partially inhibited taxol-induced apoptosis, whereas the caspase-10 inhibitor totally abrogated this process. Taxol-induced apoptosis was also associated with decreased mitochondrial membrane potential (Deltapsim) and a significant increase in ROS generation. However, increased ROS production was not directly involved in taxol-triggered apoptosis. Therefore, these results demonstrate for the first time that taxol induces FADD-dependent apoptosis primarily through activation of caspase-10 but independently of death receptors.