Acute denervation of the lungs occurs after heart-lung transplantation (HLT), affecting both afferent and efferent nerves below the tracheal anastomosis. After surgery, the carina and main bronchi are perfused by mediastinal collaterals derived from the coronary arteries, and the intrapulmonary airways by retrograde blood flow from pulmonary artery collaterals. During acute rejection, the lungs are subjected to inflammation, particularly perivascular lymphocytic infiltrates. Rejection can be diagnosed by transbronchial biopsy (TBB). We report the bronchial responses to inhaled methacholine and ultrasonically nebulized distilled water (USNDW) in 16 HLT patients 2 wk to 43 months after surgery, relating them to the lung histopathology from concurrent TBB. Methacholine bronchial hyperresponsiveness was common, but it was not associated with airway epithelial or submucosal inflammation or perivascular lymphocytic infiltration. Six patients had a modest response to USNDW (fall in FEV1 greater than 10%). The responsiveness to USNDW was not associated with enhanced methacholine responsiveness or epithelial and mucosal inflammation. However, it was more commonly seen in patients with lung rejection and perivascular infiltrates. Methacholine hyperresponsiveness in HLT patients could therefore reflect denervation hypersensitivity of airway smooth muscle muscarinic receptors. The modest response to USNDW in some patients cannot be a result of a vagal reflex but could reflect a pathologic vascular response associated with lung rejection. These observations offer insight into the possible mechanisms of bronchial hyperresponsiveness in disease.

ABSACTThe main clinical problems that follow heart-lung transplantation are opportunist infections of the lungs and pulmonary rejection. Of 23 patients undergoing heart-lung transplantation, eight had opportunist infections and 12 had at least one episode ofpulmonary rejection. Cardiac rejection occurred in only one patient, who did not need treatment. Of the 12 patients who had pulmonary rejection, nine recovered fully after augmented immunosuppression with high dose corticosteroids, although one patient required additional low dose corticosteroids for eight months before making a full recovery. Fatal opportunist lung infection followed treatment for rejection in two patients. One patient developed obliterative bronchiolitis. Of the eight patients with opportunist infections, five had primary cytomegalovirus pneumonitis, acquired from the donor. All three patients treated with acyclovir died, whereas the two treated with hyperimmune globulin and dihydroxy proxymethylguanine recovered fully. Two patients developed Pneumoncystis carinii pneumonia, which was treated successfully in one patient with intravenous sulphadimidine and trimethoprim. The other patient died after a further episode of rejection and aspergillus bronchitis. One patient developed a tuberculous empyema. The calculated actuarial survival at one year was 78% and at two years 67-2%. Although it is still in its innovative stage heart-lung transplantation appears to have complications and results similar to those of transplantation of other organs.

DNA single-strand (ss) breaks were detected in the livers of B6C3F1 mice immediately following exposure to 4000-8000 p.p.m. methylene chloride (MC) for 6 h. This damage was undetectable 2 h after exposure, suggesting an active DNA repair process. Similarly, DNA ss breaks were detected in whole lung homogenates taken from mice exposed to 2000-6000 p.p.m. MC. The DNA of mouse Clara cells incubated in vitro with MC was also damaged at concentrations of 5 mM MC and above. Pre-treatment of mice with the glutathione depletor buthionine sulphoximine (BSO) caused a decrease in the amount of DNA damage detected, suggesting a GST-mediated mechanism. DNA damage was also reduced in Clara cells when incubated in vitro with MC in the presence of BSO. In CHO cells induction of DNA damage was dependent upon exogenous MC metabolism by mouse liver S100 fraction (but not microsomes) in the presence of GSH. DNA ss breaks were not induced by MC in hamster hepatocytes in vitro at concentrations from 5 to 90 mM MC, nor in eight individual samples of normal human hepatocytes exposed to MC at similar concentrations. The ability of MC to induce DNA ss breaks in the four species studied is entirely compatible with the known carcinogenicity of this chemical in animals and offers experimental evidence to suggest that humans would not be susceptible to MC-induced liver cancer. The DNA ss breaks correlate with the metabolism of MC by the GST pathway and provide an explanation for the lack of sensitivity of hamsters and rats to MC-induced liver cancer.

Trichloroacetic acid (TCA) was tested in mice for its ability to cause single-strand breaks (SSBs) in hepatic DNA in the presence and absence of liver growth induction. Male B6C3F1 mice were given 1, 2 or 3 daily doses of TCA (500 mg/kg p.o.) as a neutralized solution (sodium salt) and killed 1 h after the final dose. Some mice were given a single dose of TCA (500 mg/kg p.o.) as the acid or as a neutralized solution and killed 24 h after. Liver nuclei were prepared and the induction of DNA SSBs assayed. TCA gave no significant response. Absorption and distribution studies were conducted with radiolabelled trichloro[2-14C]acetic acid, which was administered by gavage (500 mg/kg) as aqueous free acid, neutral aqueous solution (sodium salt) or free acid in corn oil. The absorption and distribution of TCA was similar in all cases: the chemical was absorbed rapidly after dosing, maximum plasma and liver concentrations of free radiolabel being achieved in less than 1 h. Within the first 4 h following dosing there was no evidence of binding to DNA or other macromolecules in plasma and very little 'covalent' binding was detected in liver, indicating that at times when maximum DNA single-strand breakage has been reported there was no significant binding to liver cells. Studies on liver growth parameters (hyperplasia and peroxisome proliferation) with TCA revealed that the chemical induced small but significant increases in both parameters. No SSB induction was detected in association with either liver growth phenomenon elicited by TCA. We have thus found no evidence that TCA causes SSBs in the hepatic DNA of treated mice, in contrast to previous observations by other investigators.

Methylene chloride (MC) induced DNA damage in freshly isolated hepatocytes from mice and rats, which was detectable as single-strand (ss) breaks by alkaline elution. The lowest in vitro concentration of MC needed to induce DNA damage in mouse hepatocytes (0.4 mM) was much lower than for rat hepatocytes (30 mM), and is close to the calculated steady-state concentration of MC in the mouse liver (1.6 mM) at a carcinogenic dose (4000 p.p.m. by inhalation). DNA ss breaks were also detectable in hepatocyte DNA from mice which had inhaled 4000 p.p.m. MC for 6 h, but not in hepatocyte DNA from rats similarly exposed. In studies with hepatocytes cultured overnight in the presence of buthionine sulfoximine to deplete glutathione (GSH), subsequent exposure to MC resulted in less DNA damage in the GSH-depleted cells. This shows that conjugation of MC with GSH is important in its activation of DNA-damaging species in the liver. The GSH pathway of MC metabolism produces two potential DNA-damaging species, formaldehyde and S-chloromethylglutathione (GSCH2Cl). Formaldehyde is known to cause DNA ss breaks in cells. However, the lowest concentration of formaldehyde required to induce a significant amount of DNA ss breaks in mouse hepatocytes (0.25 mM) is unlikely to be formed following in vitro or in vivo metabolism of MC at concentrations that induce similar amounts of DNA damage. That formaldehyde does not play a role in this DNA damage has been confirmed in experiments with CHO cells exposed to MC and an exogenous activation system from mouse liver (S9 fraction). Formaldehyde was responsible for the DNA- protein cross-linking effect of MC, but did not cause the DNA damage leading to ss breaks. These DNA ss breaks are likely to be caused by GSCH2Cl. The results suggest a genotoxic mechanism for MC carcinogenicity in the mouse liver, and support the proposal that the observed species differences in liver carcinogenicity result from differences in the amount of MC metabolism via the GSH pathway in the target organ.