Diamond Quantum Sensing Revealing the Relation between Free Radicals and Huntington's Disease.

Huntington's disease (HD) is a well-studied yet rare disease caused by a specific mutation that results in the expression of polyglutamine (PolyQ). The formation of aggregates of PolyQ leads to disease and increases the level of free radicals. However, it is unclear where free radicals are generated and how they impact cells. To address this, a new method called relaxometry was used to perform nanoscale MRI measurements with a subcellular resolution. The method uses a defect in fluorescent nanodiamond (FND) that changes its optical properties based on its magnetic surroundings, allowing for sensitive detection of free radicals. To investigate if radical generation occurs near PolyQ aggregates, stable tetracycline (tet)-inducible HDQ119-EGFP-expressing human embryonic kidney cells (HEK PQ) were used to induce the PolyQ formation and Huntington aggregation. The study found that NDs are highly colocalized with PolyQ aggregates at autolysosomes, and as the amount of PolyQ aggregation increased, so did the production of free radicals, indicating a relationship between PolyQ aggregation and autolysosome dysfunction.

Polyglutamine aggregation in Huntington's disease and spinocerebellar ataxia type 3: similar mechanisms in aggregate formation.

AIMS: Polyglutamine (polyQ) diseases are characterized by the expansion of a polymorphic glutamine sequence in disease-specific proteins and exhibit aggregation of these proteins. This is combated by the cellular protein quality control (PQC) system, consisting of chaperone-mediated refolding as well as proteasomal and lysosomal degradation pathways. Our recent study in the polyQ disease spinocerebellar ataxia type 3 (SCA3) suggested a distinct pattern of protein aggregation and PQC dysregulation. METHODS: To corroborate these findings we have investigated immunohistochemically stained 5 µm sections from different brain areas of Huntington's disease (HD) and SCA3 patients. RESULTS: Irrespective of disease and brain region, we observed peri- and intranuclear polyQ protein aggregates. A subset of neurones with intranuclear inclusions bodies exhibited signs of proteasomal dysfunction, up-regulation of HSPA1A and re-distribution of DNAJB1. The extent of the observed effects varied depending on brain area and disease protein. CONCLUSIONS: Our results suggest a common sequence, in which formation of cytoplasmic and nuclear inclusions precede proteasomal impairment and induction of the cellular stress response. Clearly, impairment of the PQC is not the primary cause for inclusion formation, but rather a consequence that might contribute to neuronal dysfunction and death. Notably, the inclusion pathology is not directly correlated to the severity of the degeneration in different areas, implying that different populations of neurones respond to polyQ aggregation with varying efficacy and that protein aggregation outside the neuronal perikaryon (e.g. axonal aggregates) or other effects of polyQ aggregation, which are more difficult to visualize, may contribute to neurodegeneration.

The small heat shock proteins family: the long forgotten chaperones.

Small heat shock proteins are a rather heterogeneous family of ATP-independent chaperones, some of which have been proven to block protein aggregation and help the cells to survive stressful conditions. Although much less studied than high molecular weight HSPs like HSP70/HSPA or HSP90/HSPC, their implication in physio-pathological processes and human diseases is now well evidenced, as it will be discussed in the different reviews of this special issue. In this mini-review we will just present a general introduction about the small heat shock proteins family. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

The HSPB8-BAG3 chaperone complex is upregulated in astrocytes in the human brain affected by protein aggregation diseases.

AIMS: HSPB8 is a small heat shock protein that forms a complex with the co-chaperone BAG3. Overexpression of the HSPB8-BAG3 complex in cells stimulates autophagy and facilitates the clearance of mutated aggregation-prone proteins, whose accumulation is a hallmark of many neurodegenerative disorders. HSPB8-BAG3 could thus play a protective role in protein aggregation diseases and might be specifically upregulated in response to aggregate-prone protein-mediated toxicity. Here we analysed HSPB8-BAG3 expression levels in post-mortem human brain tissue from patients suffering of the following protein conformation disorders: Alzheimer's disease, Parkinson's disease, Huntington's disease and spinocerebellar ataxia type 3 (SCA3). METHODS: Western blotting and immunohistochemistry techniques were used to analyse HSPB8 and BAG3 expression levels in fibroblasts from SCA3 patients and post-mortem brain tissues, respectively. RESULTS: In all diseases investigated, we observed a strong upregulation of HSPB8 and a moderate upregulation of BAG3 specifically in astrocytes in the cerebral areas affected by neuronal damage and degeneration. Intriguingly, no significant change in the HSPB8-BAG3 expression levels was observed within neurones, irrespective of their localization or of the presence of proteinaceous aggregates. CONCLUSIONS: We propose that the upregulation of HSPB8 and BAG3 may enhance the ability of astrocytes to clear aggregated proteins released from neurones and cellular debris, maintain the local tissue homeostasis and/or participate in the cytoskeletal remodelling that astrocytes undergo during astrogliosis.

Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3).

AIMS: A characteristic of polyglutamine diseases is the increased propensity of disease proteins to aggregate, which is thought to be a major contributing factor to the underlying neurodegeneration. Healthy cells contain mechanisms for handling protein damage, the protein quality control, which must be impaired or inefficient to permit proteotoxicity under pathological conditions. METHODS: We used a quantitative analysis of immunohistochemistry of the pons of eight patients with the polyglutamine disorder spinocerebellar ataxia type 3. We employed the anti-polyglutamine antibody 1C2, antibodies against p62 that is involved in delivering ubiquitinated protein aggregates to autophagosomes, antibodies against the chaperones HSPA1A and DNAJB1 and the proteasomal stress marker UBB⁺¹. RESULTS: The 1C2 antibody stained neuronal nuclear inclusions (NNIs), diffuse nuclear staining (DNS), granular cytoplasmic staining (GCS) and combinations, with reproducible distribution. P62 always co-localized with 1C2 in NNI. DNS and GCS co-stained with a lower frequency. UBB⁺¹ was present in a subset of neurones with NNI. A subset of UBB⁺¹-containing neurones displayed increased levels of HSPA1A, while DNAJB1 was sequestered into the NNI. CONCLUSION: Based on our results, we propose a model for the aggregation-associated pathology of spinocerebellar ataxia type 3: GCS and DNS aggregation likely represents early stages of pathology, which progresses towards formation of p62-positive NNI. A fraction of NNI exhibits UBB⁺¹ staining, implying proteasomal overload at a later stage. Subsequently, the stress-inducible HSPA1A is elevated while DNAJB1 is recruited into NNIs. This indicates that the stress response is only induced late when all endogenous protein quality control systems have failed.

Levels of DNAJB family members (HSP40) correlate with disease onset in patients with spinocerebellar ataxia type 3.

In polyglutamine disorders, the length of the expanded CAG repeat shows a strong inverse correlation with the age at disease onset, yet up to 50% of the variation in age of onset is determined by other additional factors. Here, we investigated whether variations in the expression of heat shock proteins (HSP) are related to differences in the age of onset in patients with spinocerebellar ataxia (SCA)3. Hereto, we analysed the protein expression levels of HSPA1A (HSP70), HSPA8 (HSC70), DNAJB (HSP40) and HSPB1 (HSP27) in fibroblasts from patients and healthy controls. HSPB1 levels were significantly upregulated in fibroblasts from patients with SCA3, but without relation to age of onset. Exclusively for expression of DNAJB family members, a correlation was found with the age of onset independent of the length of the CAG repeat expansion. This indicates that DNAJB members might be contributors to the variation in age of onset and underlines the possible use of DNAJB proteins as therapeutic targets.

Modulation of polyglutamine inclusion formation by the Hsp70 chaperone machine.

Components of the Hsp70 chaperone machine have been implied in protection against polyglutamine (poly-Q) pathologies. Yet, little is known about specific mechanisms and the rate-limiting components that account for this protective effect. Here, we examined the effects of an Hsp70 chaperone family member (HspA1A) and its cofactors Hsp40 (DnaJB1), Bag-1 and CHIP on poly-Q protein inclusion formation and SDS-insolubilization. Overexpression of HspA1A alone did not suppress inclusion formation, while overexpression of DnaJB1 reduced poly-Q inclusion formation and insolubilization. The reducing effect of DnaJB1 on inclusion formation was enhanced by coexpressing HspA1A, and was dependent on the interaction of DnaJB1 with Hsp70/Hsc70 chaperones. Additionally, two factors connecting Hsp70 activity with protein degradation by the ubiquitin-proteasome system Bag-1 and CHIP slightly decreased the levels of soluble poly-Q protein, but the amount of aggregated protein and fraction of cells with inclusions remained unaltered. Our data suggest that the HspA1A chaperone machine can modulate poly-Q inclusion formation depending on the ratio of its components and that DnaJB1 is the rate-limiting step.

Heat shock proteins and Bcl-2 expression and function in relation to the differential hyperthermic sensitivity between leukemic and normal hematopoietic cells.

A major problem in autologous stem cell transplantation is the occurrence of relapse by residual neoplastic cells from the graft. The selective toxicity of hyperthermia toward malignant hematopoietic progenitors compared with normal bone marrow cells has been utilized in purging protocols. The underlying mechanism for this selective toxicity has remained unclear. By using normal and leukemic cell line models, we searched for molecular mechanisms underlying this selective toxicity. We found that the differential heat sensitivity could not be explained by differences in the expression or inducibility of Hsp and also not by the overall chaperone capacity of the cells. Despite an apparent similarity in initial heat-induced damage, the leukemic cells underwent heat-induced apoptosis more readily than normal hematopoietic cells. The differences in apoptosis initiation were found at or upstream of cytochrome c release from the mitochondria. Sensitivity to staurosporine-induced apoptosis was similar in all cell lines tested, indicating that the apoptotic pathways were equally functional. The higher sensitivity to heat-induced apoptosis correlated with the level of Bcl-2 protein expression. Moreover, stable overexpression of Bcl-2 protected the most heat sensitive leukemic cells against heat-induced apoptosis. Our data indicate that leukemic cells have a specifically lower threshold for heat damage to initiate and execute apoptosis, which is due to an imbalance in the expression of the Bcl-2 family proteins in favor of the proapoptotic family members.

Chaperones in preventing protein denaturation in living cells and protecting against cellular stress.

A variety of cellular internal and external stress conditions can be classified as proteotoxic stresses. Proteotoxic stresses can be defined as stresses that increase the fraction of proteins that are in an unfolded state, thereby enhancing the probability of the formation of intracellular aggregates. These aggregates, if not disposed, can lead to cell death. In response to the appearance of damaged proteins, cells induce the expression of heat shock proteins. These can function as molecular chaperones to prevent protein aggregation and to keep proteins in a state competent for either refolding or degradation. Most knowledge of the function and regulation (by co-factors) of individual heat shock proteins comes from cell free studies on refolding of heat- or chemically denatured, purified proteins. Unlike the experimental situation in a test tube, cells contain multiple chaperones and co-factors often moving in and out different subcompartments that contain a variety of protein substrates at different folding states. Also, within cells folding competes with the degradative machinery. In this chapter, an overview will be provided on how the main cytosolic/nuclear chaperone Hsp70 is regulated, what is known about its interaction with other main cytosolic/nuclear chaperone families (Hsp27, Hsp90, and Hsp110), and how it may function as a molecular chaperone in living mammalian cells to protect against proteotoxic stresses.

Continuous growth of telomerase-immortalised fibroblasts: how long do cells remain normal?

Previously, we reported successful immortalisation following hTERT introduction in primary human fibroblasts, strain VH25. Since one subclone in that study developed some abnormalities, we decided to study eight additional independent immortalised clones to get an indication of the frequency and type of abnormalities that develop after hTERT-mediated immortalisation. We show that although some cell lines can maintain a normal phenotype for 500 population doublings (PDs), in four clones after 150-300PDs changes developed in basal and radiation-induced p53 and p21(WAF-1,CIP-1) levels. Our experiments demonstrate that, after prolonged culture, cells with abnormalities in cell cycle control parameters can take over the population. This calls for caution when working with hTERT-immortalised cells in vitro as well as in vivo.

ERCC1, hRad51, and BRCA1 protein expression in relation to tumour response and survival of stage III/IV NSCLC patients treated with chemotherapy.

Aim of this explorative study was to determine the prognostic value of protein expression of the DNA damage repair enzymes ERCC1, hRad51, and BRCA1 for tumour response and survival of non-small-cell lung cancer patients treated with chemotherapy. Patients with either a short or long overall survival were selected from a randomized phase III trial comparing cisplatin-gemcitabine and epirubicin-gemcitabine. Tumour biopsies were assessed for differences in immunohistochemical staining using antibodies against ERCC1, hRad51, and BRCA1. A total of 33 patients were included. A positive nuclear staining for ERCC1, hRad51, and BRCA1 was observed in 44, 12, and 90% of biopsies, respectively. In large cell carcinoma nuclear hRad51 staining was absent. In five biopsies stained for hRad51 an unexpected membrane-like staining was observed; these biopsies showed no nuclear staining. DNA damage repair protein expressions were not significantly different in responders versus non-responders, or in patients with a short or long overall survival. In conclusion, immunohistochemical staining of ERCC1, hRad51, and BRCA1, in tumour biopsies from non-small-cell lung cancer patients was not predictive for tumour response and survival after chemotherapy.

Renal expression of heat shock proteins after brain death induction in rats.

The majority of transplanted kidneys are derived from brain-dead patients. This nonphysiological state influences the hemodynamic and hormonal status of the donor. As a result, kidneys derived from brain-dead donors have inferior graft survival and increased graft function loss. Heat shock proteins (HSPs) are a family of stress-inducible proteins involved in maintaining cell homeostasis and regulating the immune system. We studied renal expression of the genes HO-1, HSP27, HSP40, and HSP70 after experimental brain death in rats. Brain death was induced in male F344 rats by slowly inflating a balloon catheter in the epidural space. Untreated rats were used as controls. Animals were humanely killed after 4 hours of brain death. Kidneys were analysed using RT-PCR, Western blotting, and immunohistochemistry. RT-PCR showed an increase in expression of genes coding for HO-1 (3.6-fold; P < .05) and HSP70 (2.7-fold; P < .05) after brain death. Western blotting also revealed an increase in HO-1 protein levels (4.6-fold; P < .001) but changes in HSP70 protein expression were not detected. Immunohistochemistry showed increments of HO-1 protein expression in the renal cortical tubules of brain-dead rats. HSP70 was predominantly increased in renal distal tubules of brain-dead rats treated for hypotension. No changes were observed in renal HSP27 and HSP40 expression after brain death. Renal stress caused by brain death induces expression of the cytoprotective genes HO-1 and HSP70, but not of HSP27 and HSP40. The up-regulation of these cytoprotective genes could be part of a recuperative mechanism induced by stress associated with brain death.

Defects in muscarinic receptor-coupled signal transduction in isolated parotid gland cells after in vivo irradiation: evidence for a non-DNA target of radiation.

Radiation-induced dysfunction of normal tissue, an unwanted side effect of radiotherapeutic treatment of cancer, is usually considered to be caused by impaired loss of cell renewal due to sterilisation of stem cells. This implies that the onset of normal tissue damage is usually determined by tissue turnover rate. Salivary glands are a clear exception to this rule: they have slow turnover rates (>60 days), yet develop radiation-induced dysfunction within hours to days. We showed that this could not be explained by a hypersensitivity to radiation-induced apoptosis or necrosis of the differentiated cells. In fact, salivary cells are still capable of amylase secretion shortly after irradiation while at the same time water secretion seems specifically and severely impaired. Here, we demonstrate that salivary gland cells isolated after in vivo irradiation are impaired in their ability to mobilise calcium from intracellular stores (Ca2+ i), the driving force for water secretion, after exposure to muscarinic acetylcholine receptor agonists. Using radioligand-receptor-binding assays it is shown that radiation caused no changes in receptor density, receptor affinity nor in receptor-G-protein coupling. However, muscarinic acetylcholine agonist-induced activation of protein kinase C alpha (PKCalpha), measured as translocation to the plasma membrane, was severely affected in irradiated cells. Also, the phorbol ester PMA could no longer induce PKCalpha translocation in irradiated cells. Our data hence indicate that irradiation specifically interferes with PKCalpha association with membranes, leading to impairment of intracellular signalling. To the best of our knowledge, these data for the first time suggest that, the cells' capacity to respond to a receptor agonist is impaired after irradiation.

Mechanism of radiosensitization by hyperthermia (> or = 43 degrees C) as derived from studies with DNA repair defective mutant cell lines.

All biochemical and cytogenetic data on radiosensitization by heat treatment at and above 43 degrees C indicate that inhibition of DNA repair plays a central role. There are several DNA repair pathways involved in restoration of damage after ionising irradiation and the kinetics of all of them are affected by heat shock. This, however, does not imply that the inhibition of each of these pathways is relevant to the effect of heat on cellular radiosensitivity. The current review evaluates the available data on heat radiosensitization in mutant or knockout cell lines defective in various DNA repair proteins and/or pathways. The data show that thermal inhibition of the non-homologous end-joining pathway (NHEJ) plays no role in heat radiosensitization. Furthermore, limited data suggest that the homologous recombination pathway may also not be a major heat target. By deduction, it is suggested that inhibition of base damage repair (BER) could be the crucial step in radiosensitization by heat. While a lack of mutant cell lines and redundancy of the BER pathway have hampered efforts toward a conclusive study, biochemical and correlative evidence support this hypothesis.

Reconstitution of active telomerase in primary human foreskin fibroblasts: effects on proliferative characteristics and response to ionizing radiation.

PURPOSE: Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear whether cells immortalized by telomerase reactivation behave in a similar manner as their parental primary cells. MATERIALS AND METHODS: Primary human foreskin fibroblasts were transfected with the human catalytic subunit of telomerase, the reverse transcriptase (hTERT), and their growth characteristics and response to DNA damage were characterized. RESULTS: The sole expression of the human hTERT was sufficient to immortalize the human foreskin fibroblasts. With time in culture, the immortalized cells almost doubled their average telomeric length and the clonal population contained almost no post-mitotic fibroblasts anymore. Up to 300 population doublings, no alterations compared with the parental primary cells were seen in terms of clonogenic radiosensitivity, DNA double-strand break repair, radiation-induced increases in p53 and p21(WAF-1,CIP-1) expression, and the G1/S and G2/M cell cycle checkpoints. Moreover, mitogen-induced mitotic arrest of fibroblasts was still possible in the hTERT-immortalized clones. CONCLUSIONS: Immortalizing fibroblasts by reconstitution of active telomerase seems a good, reliable manner to generate a large source of cells with a radiation damage response similar to the primary cells.

Loco-regional differences in pulmonary function and density after partial rat lung irradiation.

PURPOSE: The purpose of this study was to explore regional differences in radiosensitivity of rat lung using lung function and computed tomography (CT) density as endpoints. METHODS: At first, CT scans were used to determine rat lung volumes. The data obtained enabled the design of accurate collimators to irradiate 50% of the total lung volume for the apex, base, left, right, mediastinal and lateral part of the lung. Male Wistar rats were irradiated with a single dose of 18 Gy of orthovoltage X-rays. Further rat thorax CT scans were made before and 4, 16, 26, and 52 weeks after irradiation to measure in vivo lung density changes indicative of lung damage. To evaluate overall lung function, breathing frequencies were measured biweekly starting 1 week before irradiation. RESULTS: Qualitative analysis of the CT scans showed clear density changes for all irradiated lung volumes, with the most prominent changes present in the mediastinal and left group at 26 weeks after radiation. Quantitative analysis using average density changes of whole lungs did not adequately describe the differences in radiation response between the treated groups. However, analysis of the density changes of the irradiated and non-irradiated regions of interest (ROI) more closely matched with the qualitative observations. Breathing frequencies (BF) were only increased after 50% left lung irradiation, indicating that the hypersensitivity of the mediastinal part as assessed by CT analysis, does not result in functional changes. CONCLUSIONS: For both BF and CT (best described by ROI analysis), differences in regional lung radiosensitivity were observed. The presentation of lung damage either as function loss or density changes do not necessarily coincide, meaning that for each endpoint the regional sensitivity may be different.

BRCA1 and BRCA2 heterozygosity and repair of X-ray-induced DNA damage.

PURPOSE: Up to 90% of hereditary breast cancer cases are linked to germ-line mutations in one of the two copies of the BRCA1 or BRCA2 genes. Brca1 and Brca2 proteins are both involved in the cellular defence against DNA damage, although the precise function of the proteins is still not known. Some studies on a small number of samples as well as the present pilot study also suggested that BRCA1 heterozygosity may lead to impaired repair of ionizing-radiation-induced DNA double-strand breaks. The purpose of the study was to test in a larger family-matched study whether carriers of BRCA1 or BRCA2 mutations have an increased sensitivity to ionizing radiation. MATERIALS AND METHODS: In a blind study, the effect of different germ-line mutations in one allele of the BRCA1 or BRCA2 gene on the ability to repair X-ray-induced DNA breaks was investigated. Fibroblasts and lymphocytes were taken from heterozygotic individuals (BRCA1+ /- and BRCA2+ /-) with different mutations and from relatives proven to be non-carriers of the BRCA mutations. Rejoining of DNA breaks was analysed by pulsed-field gel electrophoresis (for fibroblasts) or the comet assay (for lymphocytes). RESULTS: Significant interindividual differences were found in the capacities of the fibroblasts and lymphocytes to rejoin DNA breaks induced by X-radiation. However, these differences were not related to heterozygosity in BRCA1 or BRCA2. CONCLUSIONS: Cells from carriers of mutations in one allele of the BRCA1 or BRCA2 genes have no gross defects in their ability to rejoin radiation-induced DNA breaks. Hence, these carriers may not be at risk of developing excess normal tissue reactions after radiotherapy consistent with data from recent clinical studies.

Salivary gland-sparing prophylactic pilocarpine treatment has no effect on tumor regrowth after irradiation.

Radiotherapy of head and neck cancer frequently damages the salivary glands. Prophylactic administration of the muscarinic receptor agonist pilocarpine reduces subsequent radiation damage to the salivary glands in rats, but its effects on tumor cell radiosensitivity and tumor regrowth after irradiation had not been assessed. In the current study, we first tested the effect of pilocarpine on clonogenic cell survival in vitro. No effect of pilocarpine on radiosensitivity was observed in a panel of cell lines either with or without expression of muscarinic receptors. Second, a single dose of pilocarpine known to protect salivary gland tissue from radiation damage was given to rats transplanted with subcutaneously growing rhabdomyosarcomas 1 h prior to irradiation with a single dose of 35 Gy. No alterations in growth delay were detected (26 +/- 2 days for controls compared to 26 +/- 2 days for pilocarpine treatment). Our data indicate that pilocarpine pretreatment, which has been shown previously to protect salivary glands from radiation, does not protect tumor cells or tumors. Use of this drug therefore may lead to therapeutic gain in the treatment of head and neck cancer.

Chemo- and radiosensitivity testing in a patient with ataxia telangiectasia and Hodgkin disease.

Treatment of Hodgkin disease (HD) in ataxia telangiectasia (AT) patients is hampered by hypersensitivity to radiation and chemotherapy. Most patients die, due to toxicity or, rarely, to progressive disease. The authors report on a 9-year-old girl with stage IIA HD and AT She was treated with a tailored combined modality approach. No unacceptable toxicity was found, but the girl died of a relapse outside the irradiation field. In comparison with fibroblasts of non-AT patients, the fibroblasts of the patient were 3 times as sensitive for radiotherapy but just 1.2 times as sensitive for doxorubicin. A good correlation was shown between in vitro radio- and chemosensitivity testing and the observed clinical toxicity. The authors suggest, therefore, treating AT patients as much as possible according to standard protocols by adjusting the radiotherapy delivery and the chemotherapy regimen to individual doses derived from in vitro radio- and chemosensitivity testing.

Early to late sparing of radiation damage to the parotid gland by adrenergic and muscarinic receptor agonists.

Damage to salivary glands after radiotherapeutic treatment of head and neck tumours can severely impair the quality of life of the patients. In the current study we have investigated the early-to-late pathogenesis of the parotid gland after radiation. Also the ability to ameliorate the damage using pretreatment with adrenergic or muscarinic receptor agonists is studied. Rats were locally irradiated with or without i.p. pretreatment with phenylephrine (alpha-adrenoceptor agonist, 5 mg kg(-1)), isoproterenol (beta-adrenoceptor agonist, 5 mg kg(-1)), pilocarpine (4 mg kg(-1)), methacholine (3.75 mg kg(-1)) (muscarinic receptor agonists) or methacholine plus phenylephrine. Parotid salivary flow rate, amylase secretion, the number of cells and gland histology were monitored sequentially up to 240 days postirradiation. The effects were described in 4 distinct phases. The first phase (0-10 days) was characterised by a rapid decline in flow rate without changes in amylase secretion or acinar cell number. The second phase (10-60 days) consists of a decrease in amylase secretion and is paralleled by acinar cell loss. Flow rate, amylase secretion and acinar cell numbers do not change in the third phase (60-120 days). The fourth phase (120-240 days) is determined by a further deterioration of gland function but an increase in acinar cell number, albeit with poor tissue morphology. All drug pretreatments used could reduce radiation effects in phase I and II. The protective effects were lost during phase IV, with the exception of methacholine plus phenylephrine pretreatment. The latter combination of drugs ameliorated radiation-damage throughout the entire follow-up time. The data show that combined pre-irradiation stimulation of muscarinic acetylcholine receptors with methacholine plus alpha-adrenoceptors with phenylephrine can reduce both early and late damage, possibly involving the PLC/PIP2 second messenger pathways. This opens perspectives for the development of clinical applicable methods for long-term sparing of parotid glands subjected to radiotherapy of head and neck cancer patients.