**7. Feverfew PFE increases expression of DNA repair enzymes**

Skin is continously exposed to numerous aggressors that can cause oxidative DNA damage. The age-related accumulation of somatic damage is worsened by sun exposure, leading to an increased incidence of skin disorders and skin cancer. Chemical entities such as O2·, H2O2, OH· are also generated in cells as a result of several endogenous processes including normal cellular metabolism and mitochondrial respiration (Verjat et al., 2000). Some of these chemical species are highly reactive and can interact with DNA, lipids and proteins (Ames and Shigenaga, 1992) causing damage. Oxidative DNA damage can arise through the direct interaction of reactive species with genomic DNA, or via oxidation of DNA precursors in the nucleotide pool (Rai, 2010). One of the major DNA oxidation products formed as a result of such damage is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG). Mammalian cells have evolved several DNA-repair pathways to remove all the categories of DNA base lesions, relying in particular on DNA excision mechanisms. One of these, nucleotide excision repair, removes bulky adducts and is thus an essential mechanism for correcting UV-induced DNA damage (Sarasin, 1999). The base excision repair pathway corrects small base modifications such as oxidized and alkylated bases (Almeida and Sobol, 2007). The importance of repair mechanisms is demonstrated by the hazardous consequences of genetic defects in DNA repair (Friedberg, 2001).

Feverfew PFE increases the enzymatic activity of DNA repair enzymes in human epidermal keratinocytes. Feverfew PFE directly induced DNA repair via the nucleotide excision repair process resulting in the repair of CPD damage induced by UV exposure. Feverfew PFE also induced the repair of DNA caused by exposure to agents that produce oxidative damage in skin. The chemotherapeutic agent, Cisplatin, and the photodynamic

The downstream functional effects of Nrf2/ARE activation by Feverfew PFE were analyzed by using an Oxidative Stress and Antioxidant Defense PCR array. Human epidermal skin equivalents treated with Feverfew PFE for 24 hr were analyzed using the PCR array. Feverfew PFE led to more than 2-fold increase in the expression of several genes involved in antioxidant defense and oxidative stress. These genes included Lactoperoxidase, an antioxidant-related gene which functions as a natural antibacterial agent, Glutathione Peroxidase which functions to protect the organism from oxidative damage by reducing free hydrogen peroxide to water, inducible Nitric Oxide Synthase-2 involved in superoxide metabolism, and G protein-coupled receptor-156 which is implicated in oxidative stress

**Gene Fold-increase in Feverfew PFE-**

Skin is continously exposed to numerous aggressors that can cause oxidative DNA damage. The age-related accumulation of somatic damage is worsened by sun exposure, leading to an increased incidence of skin disorders and skin cancer. Chemical entities such as O2·, H2O2, OH· are also generated in cells as a result of several endogenous processes including normal cellular metabolism and mitochondrial respiration (Verjat et al., 2000). Some of these chemical species are highly reactive and can interact with DNA, lipids and proteins (Ames and Shigenaga, 1992) causing damage. Oxidative DNA damage can arise through the direct interaction of reactive species with genomic DNA, or via oxidation of DNA precursors in the nucleotide pool (Rai, 2010). One of the major DNA oxidation products formed as a result of such damage is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG). Mammalian cells have evolved several DNA-repair pathways to remove all the categories of DNA base lesions, relying in particular on DNA excision mechanisms. One of these, nucleotide excision repair, removes bulky adducts and is thus an essential mechanism for correcting UV-induced DNA damage (Sarasin, 1999). The base excision repair pathway corrects small base modifications such as oxidized and alkylated bases (Almeida and Sobol, 2007). The importance of repair mechanisms is demonstrated by the hazardous consequences of genetic defects in DNA

Feverfew PFE increases the enzymatic activity of DNA repair enzymes in human epidermal keratinocytes. Feverfew PFE directly induced DNA repair via the nucleotide excision repair process resulting in the repair of CPD damage induced by UV exposure. Feverfew PFE also induced the repair of DNA caused by exposure to agents that produce oxidative damage in skin. The chemotherapeutic agent, Cisplatin, and the photodynamic

Lactoperoxidase 2 Glutathione Peroxidase 2 Inducible-Nitric Oxide synthase-2 (iNOS2) 3 G-protein coupled Receptor-156 (GPR156) 3

Table 1. Fevefew PFE leads to induction of antioxidant defense genes.

**7. Feverfew PFE increases expression of DNA repair enzymes** 

**treated sample over vehicle control** 

response.

repair (Friedberg, 2001).

agent, Psoralen both induce oxidative stress which can produce DNA damage (Martin et al., 2008). Feverfew PFE induced DNA repair activity in DNA damaged by Cisplatin or Psoralen. Thus Feverfew PFE can activate the endogenous nucleotide excision repair in human epidermal keratinocytes, which could repair DNA damaged by extrinsic or intrinsic ROS formation.

In addition to the direct effects of Feverfew PFE on DNA repair enzymes, activation of the ARE/Nrf2 pathway by Feverfew might also play a crucial role in preventing DNA damage and augmenting DNA repair. Daily exposure to UV, smoke, and other external aggressors leads to the generation of high levels of reactive oxygen species in the body. In order to avoid the deleterious effects of reactive oxidative species, cells have developed different defensive mechanisms including antioxidant molecules such as glutathione, alphatocopherol, vitamins A and C, and enzymes such as superoxide dismutase (SOD) and catalase (Ames et al., 1993; Verjat et al., 2000). When the cellular balance between antioxidants and pro-oxidants is upset, free radicals lead to the formation of lesions in the DNA causing damage, which in most cases is endogenously repaired in the cells by several repair mechanisms. The Nrf2/ARE pathway induces production of a battery of downstream genes that mitigate damage from reactive oxygen species, and aid DNA repair (Villeneuve et al., 2009). It has been shown that activation of Nrf2 generates a glutathione gradient to protect skin from the damaging effects of UVB (Saw et al., Schafer et al.), and reduces UVAinduced apoptosis in fibroblasts (Hirota et al., 2005). Thus, Feverfew PFE may help protect the skin from DNA damage incurred by UV and other environmental aggressors by boosting the cellular antioxidant defense machinery.
