Polyamines as biochemical indicators of radiation injury.

The search for parameters of different nature to quantify radiation damage is carrying on from many years in humans and lab animals. The polyamines (spermidine and spermine) are ubiquitous polycations with many metabolic functions and can be easily assayed by HPLC method. Their involvement in cell proliferation has been evidenced in healthy and tumour tissues. Statistically significant reductions have been demonstrated in tissues and in red blood cells (RBC), in animals and in patients treated by total body irradiation (TBI) before bone marrow transplantation (BMT). In rats submitted to TBI with 3 Gy of gamma radiations, tissue polyamines significantly decrease during the early phase of injury in tissues with high proliferative activity (small intestine, spleen) whereas do not show any modification in kidney. When recovery takes place, the polyamines significantly increase and return to control levels when a normal morphology is restored. In patients submitted to radiation therapy, polyamines have been determined in urine and in RBC of patients with carcinoma of uterine cervix, head and neck and prostate, treated by external radiotherapy, and with thyroid cancer treated with iodine-131 therapy. The most interesting results has been obtained with RBC: in patients treated on the pelvis for prostate cancer a significant reduction during radiotherapy occurs, followed by the maintenance of low levels in patients with favourable outcome. It should be noted that polyamine levels before treatment appeared significantly higher than in healthy controls. After TBI the RBC polyamines show a dramatic fall to extremely low levels during the phase of marrow aplasia. The values show an increase corresponding to the engraftment of transplanted cells and to the following marrow repopulation. These evidences make the RBC polyamines very interesting parameters to monitor the radiation effects on humans.

Proposal for biochemical dosimeter for prolonged space flights.

Radiation dosimetry has been developed by means of physical, chemical and biological methods. A different approach to calculate the absorbed dose is related to the assay in body fluids of some molecules that modify their concentration after irradiation. The salivary glands in humans appear particularly radiosensitive and the effects of ionizing radiation can be evaluated by means of the determination of serum amylase (produced by acinar cells) and Tissue Polypeptide Antigen (TPA, synthesized by ductal cells). Patients submitted to external radiotherapy for tumours localized in the head and neck region show early and late effects on salivary glands. The modification of amylase activity and TPA appear as a progressive statistically significant increase within two days. Levels of 200-300% of baseline value are reached, followed by a rapid return to preirradiation levels. The use of different doses per fraction and fractionation schedules (conventional or multiple daily fractionations) confirm the direct correlation between the absorbed dose and serum amylase and TPA levels. It is worth noting that the irradiation of pancreas region did not produce any effect on amylase activity. The correlation may be assumed as linear for a short dose range (2-6 Gy) whereas in the range from 0.5 to 10 Gy a sigmoid curve represents the experimental data. Both molecules confirm their capability to quantify the absorbed dose in patients with thyroid carcinoma submitted to metabolic treatment with iodine-131. The effects of radiation are species-specific and are absent in laboratory mammals. The easiness of the determination of serum amylase and TPA lead us to propose the test as biochemical dosimeter for cosmic rays exposure during prolonged staying in the space.

Serum amylase and tissue polypeptide antigen as biochemical indicators of salivary gland injury during iodine-131 therapy.

The study evaluated the possibility of using serum alpha-amylase and tissue polypeptide antigen (TPA) as biochemical markers of radiation injury in the salivary gland of patients with thyroid carcinoma treated with iodine-131. The results demonstrated that the two molecules increased 1 day after 131I administration and returned to near control values on the 3rd day. The increase was greater and longer lasting in those patients treated with the higher 131I administered activity. However, when the patients were divided into groups based on recovery time, which determined patients with different radionuclide elimination rates, the increase in the two molecules was greater and more lasting in those subjects with a longer period of hospitalization. A comparison with the values obtained from patients with tumours of the head and neck treated with external radiotherapy demonstrated that after the 1st day of treatment the salivary gland received an absorbed dose which, based on alpha-amylase levels, ranged between 0.24 and 1.89 Gy and, based on TPA levels, between 0.28 and 2.29 Gy.

Marker determination for response monitoring: radiotherapy and disappearance curves.

This paper reports the results of studies on the possible role of biochemical markers in monitoring the effects of ionizing radiations and in the follow-up of cancer patients submitted to radiotherapy. Three different case series were analyzed: patients with head and neck cancer, prostate carcinoma and residual thyroid tumors or uptaking metastases (131-Iodine therapy). Serum TPA and amylase were serially determined in patients with head and neck or thyroid cancer to measure the radiation damage to the salivary glands. In the former group a statistically significant correlation between the increase of both molecules and the total dose administered after the first day of treatment (2, 3, 4 or 6 Gy) was observed. In patients treated for thyroid cancer the damage to the salivary glands was revealed by an increase in TPA and amylase serum levels, dependent on the dose of 131-Iodine administered. Moreover, an association was demonstrated between pretreatment values of TPA in patients with head and neck tumors and prognosis: patients with values below the cutoff have significantly higher survival rates than those with higher values. In patients with prostate carcinoma PSA was confirmed to have better diagnostic and prognostic value than PAP. Patients with metastases show an inversion or lack of negative trend in PSA levels observed in the disease-free patients. This precedes the clinical diagnosis of metastases by 1 to 15 months.

Prognostic significance of tissue polypeptide antigen (TPA) in head and neck carcinomas.

The serum tissue polypeptide antigen (TPA) was determined before and at the end of radiotherapy in a group of patients with head and neck epidermoid carcinoma. The baseline values were significantly higher than those in healthy control subjects. They did not depend on the site of the neoplasia but increased with clinical stage. A cut-off value of 85 U/l discriminated two groups of patients according to survival: patients whose values were lower than the cut-off had a disease-free 6-year survival rate of 61%, compared to only 22% in patients with higher TPA levels. Until the second year of follow-up, patients with complete response had baseline values significantly lower than relapsed patients. However, at 5 years the pretreatment values were similar for patients being disease-free, patients with recurrence and patients deceased within the 2-5 year period.

Post-irradiation hyperamylasemia as a biological dosimeter.

Serum alpha-amylase was measured before and 24 h after either total body (31 patients) or localized irradiation including the salivary glands (40 patients) or the pancreatic area (22 patients). A significant increase in amylasemia was observed for doses to the parotid glands larger than 0.5 Gy. A sigmoid function of dose was fitted to the data and predicted a maximum amylasemia level for doses larger than 4 Gy and smaller than 10 Gy. The raw data from other published series were adequately described by the same model. However, the confidence limits of the parameters remained wide, because of a considerable interindividual variability. Post-irradiation hyperamylasemia appears to provide a good criterion for triage of accidentally irradiated patients: 24 h after a dose larger than 2 Gy to the parotid glands, 91% of the patients had an amylasemia level higher than 2.5-fold the upper normal value (sensitivity). Conversely, 96% had their serum amylasemia lower than 2.5-fold the upper normal value when dose was smaller than 2 Gy (specificity). However, a retrospective estimation of the absorbed dose (dosimetry) is not likely to be very precise because of the large interindividual variability.

Cell kinetics and biochemical parameters in breast cancer.

The study analyzes biochemical and cell kinetic parameters to characterize solid tumor growth in humans. The concentrations of polyamines, CEA, the thymidine labeling index (T.L.I.) and the mitotic index (M.I.) were determined on fragments of neoplastic tissue from 18 patients with breast carcinoma. Urinary polyamines were evaluated in the same patients. Two groups of patients were distinguished according to the median value of the with high T.L.I., M.I. and tissue polyamines were significantly higher than in the group with low T.L.I., whereas tissue CEA was lower, though in a not statistically significant way. Urinary polyamines showed no variations between groups. These preliminary results showed that T.L.I. levels were higher in patients who relapsed during a 4-year follow-up than in patients achieving complete remission and remaining disease free. Results concerning polyamine concentration showed that the tissue polyamine level in breast carcinoma indicated proliferative activity, but this does not seem to be valuable for current prognostic purposes.

Urinary polyamines in patients with advanced cervical cancer or pelvic cancer recurrence during and after radiotherapy.

The urinary excretion of polyamines was evaluated before, during and after radiotherapy in 16 patients with advanced squamous cell carcinoma of the uterine cervix (stage IIb or IIIb) and in 7 cases with pelvic recurrence after surgery for various types of carcinoma. The concentration of spermidine was significantly higher in the patients with primary tumors than in those with recurrent tumors. After the first radiation fractions putrescine increased in the patients with primary tumors whereas it decreased in patients with recurrent tumors. The values tended to return to baseline levels with time following treatment initiation. Polyamine increased markedly during treatment in patients who remained disease-free for at least 5 years but not in the patients with progressive disease or relapse. The results suggest a different polyamine metabolism and a different response to radiotherapy of recurrent tumors compared to primary tumors. The increase of urinary polyamines, but not the baseline values, seemed to be correlated to the response after radiotherapy.

Polyamine levels in healthy and tumor tissues of patients with colon adenocarcinoma.

Tissue polyamine levels were determined in patients with colon adenocarcinoma to try to identify biochemical indicators able to characterize the growth and the metabolism of human solid tumors. Polyamine content was determined in the tumor and in the "healthy" mucosa sampled at different distances within the resection edges. For each patient the polyamine content in the tumor was compared with that in the mucosa. The results demonstrated that the spermidine concentration was higher in the tumor than in the healthy mucosa; the differences were statistically significant. However, spermine in the tumor increased to a lesser degree. No statistically significant differences were observed among these mucosae at different localizations, but the spermine concentration in the mucosa after the tumor showed values very close to those of the neoplasia.

Polyamines in the small intestine of rats after whole-body irradiation.

The behaviour of polyamines (putrescine, spermidine and spermine) in the small intestine was studied in rats exposed to a single whole-body dose of 3 Gy administered at four different times of the day. Polyamine content decreased a few hours after irradiation but quickly returned to normal values. Putrescine levels reached the lowest values and returned to the control levels later than spermidine and spermine. A temporary increase in spermidine and spermine content was observed from 5 to 20 days. The postirradiation behaviour of the animals exposed at different times of the day showed no important differences. Polyamine modifications were studied in relation to [3H]thymidine uptake and other morphological and biochemical parameters. The results indicate that polyamine content can be used to monitor the damage and recovery phases of radiation injury in the small intestine.

Polyamine content as a marker of radiation injury in the rat spleen.

The modifications of the polyamines putrescine (put), spermidine (spd) and spermine (spm) in rat spleen after 3 Gy whole-body irradiation were studied. Rats were irradiated at four different times of the day (00.00, 06.00, 12.00 and 18.00) and sacrificed between 12 h and 62 days after irradiation. Control animals, sacrificed at the same times of the day, showed higher levels of the spd/spm ratio during the hours of light. After irradiation the polyamine content was rapidly and significantly reduced over a period of 20 days. The modification of the amount of spm lasted for a longer period of time. Normal values of polyamine content were reached at later times when the mitotic activity was restored. The results show a close correlation between polyamine concentration and [3H]thymidine uptake.

Serum tissue polypeptide antigen (TPA): a marker of acute injury of salivary glands during radiation therapy.

The modifications of serum concentrations of TPA were monitored in patients undergoing curative radiation therapy. Patients with tumors localized in the head and neck were treated with one of four different schedules based on conventional fractionation or multiple daily fractionation where the dose per fraction and total daily dose varied. Serum TPA increased immediately on the first day of irradiation: the higher the dose, the greater the increase. These increases disappeared rapidly after the first few days of treatment. A more limited rise was observed in some cases when treatment was renewed after the first week-end split or after more prolonged interruptions. Results demonstrated that TPA is a valid biochemical marker of acute radiation injury to the salivary tissue.

Effects of irradiation with conventional and multiple daily fractionation on serum amylase activity.

The behaviour of serum alpha-amylase activity was determined in patients with head and neck cancer treated by radiation therapy. The levels of serum alpha-amylase activity during conventional and multiple daily fractionation were compared. Starting from the second day of treatment an increase of serum alpha-amylase activity was observed. The increase and its duration depended on the total daily dose: after the first day of treatment a linear correlation between serum levels and total daily dose could be observed. The size of the amylase increase correlated to the proportion of salivary gland tissue included in the irradiated volume.

Plasma amylase activity as a biochemical indicator of radiation injury to salivary glands.

Irradiation of the salivary glands produces a rapid increase of salivary amylase in serum, released by the highly radiation sensitive serous cells of the glands. Serial assays of salivary amylase in serum were performed in patients treated by radiation to the upper neck region. The changes observed were compared with the amount of salivary gland mass irradiated and with the dose fractionation modality used. The irradiated volume included either the entire salivary gland mass or less than 50 per cent of the gland. Two fractionation modalities were used: a conventional fractionation of 2 Gy per day, 5 times a week, or a multiple daily fractionation of 2 Gy, 3 times a day in two series of 4 days with a 4-day interval. Both parameters (salivary gland mass irradiated and fractionation modality used) significantly influenced the shape of the amylase curve in the serum. Serum amylase may therefore be considered a reliable biologic indicator of early injury to the salivary glands.

Dipeptidase activity in the small intestine after irradiation at different times of the day.

The behaviour of leucinaminopeptidase, an intestinal epithelium brush border enzyme, was investigated to find out whether the same irradiation does administered at different times of the day would induce differentiated modifications. Three activity phases were evidenced: increase, reduction during acute damage, and recovery. The results showed that the general behaviour in the four irradiated groups was similar, although some differences were observed mostly regarding the outset of the initial increase and the recovery. The time of irradiation affects the behaviour of the enzyme activities which show circadian oscillations. The results seem to show a quicker tendency to a return to normal values in the group irradiated at the end of the darkness period.

Unconventional dose administrations: modifications of body and small intestine weights.

The modifications of body and small intestine weight were analyzed after abdominal irradiation in rats. Different doses were administered in single sessions, in two equal fractions with different splits, in multiple daily fractionations, varying the amount of the dose and the intervals among fractions. The results show that body weight gradually decreased after irradiation and only after many days it returned to control levels. Small intestine weight decreased starting from early intervals, and a rapid increase over controls was observed at 5 days. A return to control levels was reached later. Multiple-daily-fractionation (MDF) irradiation with 12 Gy (1200 rad) produced modifications less evident that the same dose administered in single session and appeared more similar to those observed after 6 or 8 Gy, according to the different splits used. The more marked the damage the lower the interval between fractions when a total dose of 12 Gy was administered in two fractions. The modifications after 6 Gy MDF were less marked than after a single session of the same magnitude, both in body and small-intestinal weight. In conclusion the MDF used in these experiments produced a damage very easily recovered by the organism in spite of the short time of dose administration.

Post-irradiation enzyme activities of the rat small intestine: effects on circadian fluctuations.

The effects of ionizing radiations on circadian fluctuations in the activities of small bowel mucosal enzymes were studied. The fluctuations in activities of brush-border enzymes disappeared after irradiation coincidentally with changes in their activity. This change was particularly pronounced with leucinaminopeptidase. At longer periods after irradiation (50 and 74 hours) when the whole epithelium showed morphological changes, the activity of brush-border enzymes was very low. The activity of lysosomal enzymes (which do not show circadian fluctuations) increased when the epithelium showed marked morphological modifications and the stroma contained numerous inflammatory cells.

Circadian activity of rat kidney enzymes.

Alkaline phosphatase, LAP, beta-glucuronidase and cathepsin D activities and protein content of the kidney homogenate did not show any circadian rhythm in animals sacrificed at different hours of the day. The fluctuations of maltase appear modest and not dependent on a ligh/dark cycle.