In both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication in is transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases, NrdEF and NrdDG, or translesion DNA polymerases, Pol II, Pol IV, or Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, these do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was only observed under conditions where the stability of hydroxyurea was compromised and byproducts, known to damage DNA directly, were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesis, and that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell. Hydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication in before it rapidly recovers and continues in the presence of this drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further we show that hydroxyurea toxicity is only observed after toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by an inhibition of replication or depletion of deoxyribonucleotide levels in the cell.

Stevens-Johnson Syndrome (SJS) and toxic epidermal necrolysis (TEN) are Severe Cutaneous Adverse Reactions (SCARS) characterized by fever and mucocutaneous lesions leading to necrosis and sloughing of the epidermis. Conjunctival lesions are reported in 85% of patients. The pathogenesis of SJS/TEN/SCARS is not completely understood. It is hypothesized that IL-13, IL-15 and Granulysin expressed in plasma and skin may play a role. We measured the circulating levels of these cytokines in the plasma using ELISA and their expression in the skin using immunofluorescence microscopy. A total of 12 SJS/TEN skin biopsy samples (8 SJS, 2 SJS/TEN overlap and 2 TEN) were analyzed. Biopsy samples from patients with Lichen Planus (an inflammatory condition of the skin and mucous membranes) served as controls. Studies were also performed in human corneal epithelial cells where expression of these cytokines were measured following a challenge with TNF-α (0, 1, 10 and 100 ng/ml). The intensity of immunofluorescence was measured Using Imaris® software. The results showed significantly increased expression of these cytokines in the skin biopsy samples as measured by the average intensities of IL-13 (6.1 x 133.0 ± 4.231 x 10^8), and Granulysin (4.2 x 123.0 ± 4.231 x 10^8) compared to Lichen planus control (3.0 x 123.0 ±1.62 x 10^5). Increased expression of IL-13 and IL-15 were noted in cell culture studies and in the plasma samples when compared to Normal Human Plasma as controls. It is concluded that IL-13, IL-15 and Granulysin play a role in the pathogenesis of SJS/TEN.

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