**1.4 Western blot analysis**

*Free Radical Medicine and Biology*

survival [14–16].

cells from the guinea pig ileum.

Dulbecco's Modified Eagle Medium (DMEM).

**1.1 Tissues and cell culture**

**1.2 Colourimetry**

described by Yagi [26].

**1.3 Immunofluorescence analysis**

in the FL-2 channel [20, 28].

CellQuest software.

of the mitochondrial transmembrane electric potential (KΨmito) [9]. Several studies have used tumor cells to investigate the molecular pathways involved in the regulation and triggering of apoptosis by ionizing radiation (IR) [10, 11], but IR is more effective in normal than neoplasic tissue; so it is important to minimize the exposure in it and to clarify the mechanisms involved in the cellular damage [12]. In addition, damage to healthy tissues due to IR used in cancer treatment is frequently associated with the appearance of a second cancer occurring in the radiated field or in its vicinity [13]. This event could be explained by remodeling of the molecular and cellular processes triggering a number of inter- and intracellular signaling cascades that regulate the progression of the cell cycle and cell

The apoptotic pathway activated by IR is different from the extrinsic pathway activated by ligands and involves the generation of reactive oxygen species (ROS) and H2O2 [10, 17]. According to Orrenius [18], the enhanced ROS production regulates cellular metabolism, for the execution of the suicide program, by proteins released from the mitochondria. One of the factors involved in ROS-induced cell death is tumor necrosis factor alpha (TNFα) [15, 19], and mitochondria appear to participate in the production of this mediator. A number of hypotheses have been put forward to explain the mechanism by which TNFα cytotoxicity induces the intrinsic pathway [11]. Nevertheless, the mechanisms regulated by ROS is not totally clear, but our previous results described an increase in [Ca2+]i [20] and the activation of protein kinase C (PKCα and -ε) [21]. IR has not been directly demonstrated to affect proteins, including cyclins, cyclin-dependent kinases (CDKs), retinoblastoma protein (Rb), and E2F complex proteins [22–24], involved in the orchestration of the cell cycle. The goals of this study were to examine the extrinsic and intrinsic mechanisms involved in the apoptosis, and to investigate ROS and H2O2 generation and the mitochondria role under IR of intestinal smooth muscle

Fragments of the longitudinal smooth muscle layer of the guinea pig ileum (LSMLGPI) were prepared as described previously [20, 21], and the IR exposure in tissue fragments and confluent cell cultures from the LSMLGPI were exposed to single dose of 10–50 Gy, emitted by a 60Co γ-source [25]. The samples were radiated with a total dose of 10–50 Gy, and were then maintained for 3 days in

The ROS level was measured in the homogenates using the fluorescent method

The H2O2-induced lipid peroxidation (LP) was measured through the oxidation

a.The data were acquired and analyzed using a FACS Calibur flow cytometer and

b.The cell death study was measured at 585/542 nm using the log or linear model

of Fe2+ in the presence of xylenol orange in a spectrophotometer [27].

**54**

The experimental procedure was performed as previously described [21] using LSMLGPI homogenates. The following antibodies were used, anti-: caspase 3, cyclin A and cyclin B2, cyclin E, BCL-xL, BAX, cytochrome c, and BCL-2.
