Markers of Cardiotoxicity in Early Breast Cancer Patients Treated With a Hypofractionated Schedule: A Prospective Study.

PURPOSE: To evaluate, in a series of early breast cancer (BC) patients treated with hypofractionated adjuvant radiotherapy (RT), whether N-terminal-pro hormone B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin I assay measurements can predict acute clinical or preclinical cardiotoxicity. PATIENTS AND METHODS: The study comprised 44 consecutive patients, who underwent conservative surgery with or without (neo)adjuvant chemotherapy and hypofractionated adjuvant RT. The RT schedule consisted in a total dose of 42.4 Gy in 16 fractions administered 5 days per week. Twenty-one patients received a subsequent boost to the tumor bed consisting of a total dose of 10 Gy in 4 fractions delivered via a direct electron field. All patients underwent 12-lead electrocardiogram, echocardiogram, and cardiac clinical examinations before RT to assess cardiovascular risk factors; these examinations were repeated yearly for 5 consecutive years. High-sensitivity cardiac troponin I and NT-proBNP were analyzed from serum samples at baseline, after delivery of the fourth and 16th RT fractions, and 12 months after treatment completion. RESULTS: No increase in cardiac troponin I and B-type natriuretic peptide levels related to left breast irradiation was observed. No statistical difference in NT-proBNP and high-sensitivity troponin I levels between left- and right-sided BC was found. An increase was observed of B-type natriuretic peptide levels at baseline, during treatment, and until 12 months after RT related to hypertension, with the P value near to the .05 threshold for age and chemotherapy. CONCLUSION: Conformational hypofractionated RT in left-sided BC may not cause acute myocardial damage. Early cardiac screening may be used to identify patients with cardiologic risk factors, patients who are older than 60 years, and patients who received chemotherapy that could result in clinically relevant cardiac pathologies.

Low levels of cobalamin, epidermal growth factor, and normal prions in multiple sclerosis spinal cord.

We have previously demonstrated that multiple sclerosis (MS) patients have abnormal cerebrospinal fluid (CSF) levels of the key myelin-related molecules cobalamin (Cbl), epidermal growth factor (EGF), and normal cellular prions (PrP(C)s), thus confirming that some CSF abnormalities may be co-responsible for remyelination failure. We determined the levels of these three molecules in post-mortem spinal cord (SC) samples taken from MS patients and control patients. The control SC samples, almost all of which came from non-neurological patients, did not show any microscopic lesions of any type. All of the samples were supplied by the U.K. MS Tissue Bank. The Cbl, EGF, and PrP(C) levels were determined using enzyme-linked immunosorbent assays. The SC total homocysteine level was determined using a competitive immunoenzymatic assay. CSF samples, taken from a further group of MS patients, were used for the assay of holo-transcobalamin (holo-TC) levels. The Cbl, EGF, and PrP(C) levels were significantly decreased in MS SCs in comparison with controls and, paradoxically, the decreased Cbl levels were associated with decreased SC levels of homocysteine, a biochemical marker of Cbl deficiency. The trends of EGF and PrP(C) levels paralleled those previously found in CSF, whereas that of Cbl was the opposite. There was no significant difference in CSF holo-TC levels between the MS patients and the controls. Given that we have previously demonstrated that Cbl positively regulates central nervous system EGF levels, it is conceivable that the low EGF levels in the MS SC may be causally related to a local decrease in Cbl levels. Only PrP(C) levels were invariably decreased in both the SC and CSF regardless of the clinical course of the disease. These findings suggest that the simultaneous lack of Cbl, EGF, and PrP(C)s may greatly hamper the remyelination process in MS patients, because they are key molecules of the machinery for remyelination.

The multi-faceted basis of vitamin B12 (cobalamin) neurotrophism in adult central nervous system: Lessons learned from its deficiency.

Glial cells, myelin and the interstitium are the structures of the mammalian central nervous system (CNS) mainly affected by vitamin B(12) (cobalamin, Cbl) deficiency. Most of the response to the damage caused by Cbl deficiency seems to come from astrocytes and microglia, and is manifested as an increase in the number of cells positive for glial fibrillary acidic protein, the presence of ultrastructural signs of activation, and changes in cytokine and growth factor production and secretion. Myelin damage particularly affects the lamellae, which are disorganized by edema, as is the interstitium. Surprisingly, rat Schwann cells (myelin-forming cells of the peripheral nervous system) are fully activated but the few oligodendrocytes (myelin-forming cells of the CNS) are scarcely activated. The presence of intramyelin and interstitial edema raises questions about the integrity of the blood-brain barrier and blood-cerebrospinal fluid (CSF) barrier. The results obtained in the CNS of Cbl-deficient rats indicate that cytokine and growth factor imbalance is a key point in the pathogenesis of Cbl-deficient neuropathy. In the rat, Cbl deficiency increases the spinal cord (SC) synthesis and CSF levels of myelinotoxic cytokines (tumor necrosis factor (TNF)-alpha and soluble (s) CD40:sCD40 ligand dyad) and a myelinotoxic growth factor (nerve growth factor), but decreases SC synthesis and CSF levels of a myelinotrophic cytokine (interleukin-6) and a myelinotrophic growth factor (epidermal growth factor, EGF). The in vivo administration of IL-6 or EGF, or agents antagonizing the excess myelinotoxic agent, is as effective as Cbl in repairing or preventing Cbl-deficiency-induced CNS lesions. An imbalance in TNF-alpha and EGF levels has also been found in the CSF and serum of patients with severe Cbl deficiency.

Human cobalamin deficiency: alterations in serum tumour necrosis factor-alpha and epidermal growth factor.

OBJECTIVES: We have previously demonstrated that vitamin B12 (cobalamin)-deficient central neuropathy in the rat is associated with local overexpression of neurotoxic tumour necrosis factor (TNF)-alpha combined with locally decreased synthesis of neurotrophic epidermal growth factor (EGF). The aims of this study were to investigate whether a similar imbalance also occurs in the serum of adult patients with clinically confirmed cobalamin deficiency and whether it can be corrected by vitamin B12 replacement therapy. PATIENTS AND METHODS: We studied 34 adult patients with severe cobalamin deficiency, 12 patients with pure iron deficiency anaemia and 34 control subjects. Haematological markers of cobalamin deficiency and serum TNF-alpha and EGF levels were measured using commercial kits. Thirteen cobalamin-deficient patients were re-evaluated after 3 and 6 months of parenteral vitamin B12 treatment. RESULTS: TNF-alpha was significantly higher (p < 0.01) and EGF significantly lower (p < 0.01) in the patients with cobalamin deficiency, but both were unchanged in patients with pure iron deficiency anaemia. In cobalamin-deficient patients the serum TNF-alpha levels correlated significantly with plasma total homocysteine levels (r = 0.425; p < 0.02). In the treated patients TNF-alpha and EGF levels normalised concomitantly with clinical and haematological disease remission. CONCLUSIONS: In humans, as in rats, cobalamin concentration appears to be correlated with the synthesis and release of TNF-alpha and EGF in a reciprocal manner, because cobalamin deficiency is accompanied by overproduction of TNF-alpha and underproduction of EGF.

Further evidence for the involvement of epidermal growth factor in the signaling pathway of vitamin B12 (cobalamin) in the rat central nervous system.

In order to get further evidence for a mandatory involvement of epidermal growth factor (EGF) in the neutrophic action of vitamin B12 (cobalamin (Cbl)) in the central nervous system (CNS) of the rat, we observed the effects of repeated intracerebroventricular (ICV) microinjections of EGF in rats made Cbl-deficient through total gastrectomy. Morphometric analysis demonstrated a significant reduction in both intramyelinic and interstitial edema in the white matter of the spinal cord (SC) of totally gastrectomized (TGX) rats after treatment. Intramyelinic and interstitial edema are characteristic of Cbl-deficient central neuropathy in the rat. Similar lesions were also present in SC white matter of rats treated with repeated ICV microinjections of specific anti-EGF antibodies without any modification in their Cbl status. These results, together with those of a previous study showing the cessation of EGF synthesis in the CNS of TGX rats, demonstrate that: a) EGF is necessarily involved in the signaling pathway of Cbl in the rat CNS; and b) the lack of a neurotrophic growth factor EGF, and not the mere withdrawal of Cbl, causes or at least contributes to neurodegenerative Cbl-deficient central neuropathy.

Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B(12) (cobalamin) in the rat central nervous system.

We have recently demonstrated that the myelinolytic lesions in the spinal cord (SC) of rats made deficient in vitamin B(12) (cobalamin) (Cbl) through total gastrectomy (TG) are tumor necrosis factor-alpha (TNF-alpha)-mediated. We investigate whether or not permanent Cbl deficiency, induced in the rat either through TG or by chronic feeding of a Cbl-deficient diet, might modify the levels of three physiological neurotrophic factors-epidermal growth factor (EGF), vasoactive intestinal peptide (VIP), and somatostatin (SS)-in the cerebrospinal fluid (CSF) of these rats. We also investigated the ability of the central nervous system (CNS) in these Cbl-deficient rats to synthesize EGF mRNA and of the SC to take up labeled Cbl in vivo. Cbl-deficient rats, however the vitamin deficiency is induced, show a selective decrease in EGF CSF levels and an absence of EGF mRNA in neurons and glia in various CNS areas. In contrast, radiolabeled Cbl is almost exclusively taken up by the SC white matter, but to a much higher degree in totally gastrectomized (TGX) rats. Chronic administration of Cbl to TGX rats restores to normal both the EGF CSF level and EGF mRNA expression in the various CNS areas examined. This in vivo study presents the first evidence that the neurotrophic action of Cbl in the CNS of TGX rats is mediated by stimulation of the EGF synthesis in the CNS itself. It thus appears that Cbl inversely regulates the expression of EGF and TNF-alpha genes in the CNS of TGX rats.

Myelinolytic lesions in spinal cord of cobalamin-deficient rats are TNF-alpha-mediated.

Repeated intracerebroventricular (i.c.v.)microinjection of tumor necrosis factor-alpha (TNF-alpha) into normal rats causes intramyelin and interstitial edema in the white matter of the spinal cord (SC). This response is identical to that observed in the SC white matter of rats made cobalamin (Cbl) deficient by total gastrectomy (TG). Immunoblot analysis showed that: 1) the level of the biologically active form of the TNF-alpha protein (17 kDa) is higher in the SC of totally gastrectomized (TGX) rats 2 months after TG, i.e., at the postoperative time when edema is observed; 2) SC levels of TNF-alpha protein (17 kDa) in 2-mo-TGX-, Cbl-treated rats are reduced to control. Repeated i.c.v. microinjections of anti-TNF-alpha antibodies, transforming growth factor-beta1 (TGF-beta1) or interleukin-6 (IL-6) into TGX rats, begun shortly after TG, substantially reduced both intramyelin and interstitial edema in the SC white matter. This study provides the first evidence that the hallmark myelin damage of Cbl-deficient central neuropathy, which is a pure myelinolytic disease, is not caused directly by the withdrawal of the vitamin itself, but reflects enhanced production of the biologically active form of TNF-alpha by SC cells. This study thus supports the view that TGF-beta1 and IL-6 may act as neuroprotective agents in Cbl deficiency central neuropathy.

Polyneuropathy due to cobalamin deficiency in the rat.

In the present study, we investigated the peripheral nervous system (PNS) (both in terms of its ultrastructure and in terms of its function) of rats made cobalamin (Cbl)-deficient either through total gastrectomy or through prolonged feeding on a Cbl-deficient diet. In both these types of Cbl-deficient neuropathies we found: (a) ultrastructurally, intramyelin and endoneural edema, with no or minimal axonal damage in the PNS, in dorsal root ganglia, and the ventral and dorsal rootlets of the spinal cord; (b) electrophysiologically, a significant reduction in the nerve conduction velocity, consistent with that reported in (a); (c) morphometrically, a significant reduction in the density of myelinated fibers both in the sciatic nerve and in the peroneal nerve. All these pathological changes were reversed by chronic postoperative administration of Cbl into totally gastrectomized (TGX)-rats, hinting at the specificity of the damage itself in relation to the permanent Cbl-deficient status of the TGX-rats. No signs of segmental demyelination or remyelination were found. We also observed a turning of type I fibers into type II fibers in the soleus muscle of all our Cbl-deficient rats, however the Cbl deficiency had been induced. This muscular change was still present in TGX- and Cbl-treated rats, and it cannot be related to a malnutrition status, since it has been observed also in rats fed a Cbl-deficient diet. All these results demonstrate that Cbl deficiency strongly affects rat PNS within different parameters.

Subacute combined degeneration in totally gastrectomized rats: an ultrastructural study.

Severe permanent cobalamin (Cbl) deficiency was induced in rats either by total gastrectomy (TG) or through prolonged dietary Cbl deprivation. This paper deals with an ultrastructural investigation of different parts of the central nervous system (CNS) of rats made Cbl-deficient through one of these of two procedures. In both totally gastrectomized (TGX) rats and in rats chronically fed a Cbl-deficient diet, we observed intramyelin edema, with splitting of the lamellae, and interstitial edema affecting the white matter, mainly in the spinal cord (SC). These lesions were also present in the subcortical white matter, although to a lesser degree. In both TGX-rats and in rats chronically fed a Cbl-deficient diet the pyramidal tract and the optic nerve were completely spared. Vascular lesions were never observed. Intramyelin edema and interstitial edema of the white matter account for the patchy myelopathic spongy vacuolation, which is the histological hallmark of human subacute combined degeneration and has been previously seen in SC white matter of TGX-rats. Macro- and micro-glial cells in the white matter were activated, at least as seen ultrastructurally. Interestingly enough, there were activated glial cells even in the gray matter, in which neurons showed absolutely no alterations. Chronic subcutaneous Cbl administration of TGX-rats partially repaired the CNS damage. However, the amelioration produced by this treatment was greater when Cbl was given shortly after TG than when given three and four months after TG, i.e. when the lesions have already been formed.

Enhanced levels of biochemical markers for cobalamin deficiency in totally gastrectomized rats: uncoupling of the enhancement from the severity of spongy vacuolation in spinal cord.

The totally gastrectomized (TGX) rat is a new experimental model for studying the pathogenesis of cobalamin (Cbl)-deficient myelopathy, i.e., subacute combined degeneration, total gastrectomy (TG) serving as a surgical paradigm of human pernicious anemia. We determined the serum levels of some biochemical indicators of Cbl deficiency in TGX rats at 2 to 10 months after TG. Methylmalonic acid (MMA) rose within 2 months and progressively increased thereafter until the end of the investigation period. 2-Methylcitric acid (MCA) rose significantly by 6 months and showed a further increment 4 months later. Homocysteine was only clearly elevated much later than the serum MMA, i.e., 10 months after the operation. The concentrations of MMA, MCA, and cystathionine were increased in kidney, liver, and spinal cord (SC) of TGX rats at 10 months. Chronic treatment of TGX rats with Cbl greatly decreased the serum levels of all the metabolic indicators of Cbl deficiency. Chronic peroral administration of the antibiotic lincomycin to TGX rats in an attempt to suppress the enteric flora markedly decreased serum MMA levels. Only Cbl, however, given either for the first 2 months after TG or for the third and fourth postoperative months (i.e., after SC abnormalities had already appeared) significantly decreased the severity of spongy vacuolation in SC white matter, although not completely preventing or repairing the neuropathological damage. Therefore, neither the early impairment in TGX rats of the Cbl-dependent methylmalonyl-coenzyme A mutase reaction nor the more delayed impairment of the Cbl-dependent methionine synthase step, as reflected by changes in serum metabolite levels, seems to be causally related to the TG-induced spongy vacuolation in SC white matter.

Different patterns of expression of ornithine decarboxylase mRNAs in rat liver after acute administration of hepatocarcinogens.

In the present study, we have evaluated the induction of ornithine decarboxylase (ODC) activity in rat liver after acute in vivo administration of different hepatocarcinogens, and correlated the ODC activity peaks with the accumulation of the three ODC-related mRNA species in rat liver at different times after the intraperitoneal injection of different hepatocarcinogens. ODC activity peaked 16 h after 2-acetylaminofluorene (2-AAF) treatment, while accumulation of the three ODC-mRNAs, starting 4 h after the injection, was maximal 6 h later. Thioacetamide (TAA) administration caused a single peak of ODC activity 20 h after treatment, while there had been the maximum increases of the three ODC-mRNAs 4-h earlier. The first ODC activity peak occurred 20 h after treatment with 3'-methyl-4-(dimethylamino)azobenzene (MDAB), at the same time that accumulation of the ODC-mRNAs was maximum. There was no increase in ODC-mRNA accumulation at 28 h or 36 h after MDAB treatment, the time at which ODC activity once again peaked. All the ODC-related transcripts accumulated after MDAB treatment, although to different degrees. The 1.7 kilobase (kb) transcript accumulated the most after 2-AAF treatment. After TAA treatment, the 2.2 kb mRNA was the most abundantly expressed. In neonatal liver, in which ODC activity is physiologically high, the 1.7 kb mRNA is expressed more abundantly than the other two ODC-related transcripts. These results demonstrate that the peak of ODC enzyme activity does not always correspond in time with the peak of ODC-mRNA accumulation; that different hepatocarcinogens induce different patterns of accumulation of the ODC-related transcripts; and that the minor ODC-related transcript (1.7 kb) in rat liver seems to be expressed not only constitutively but is also inducible.

Subacute combined degeneration in the spinal cords of totally gastrectomized rats. Ornithine decarboxylase induction, cobalamin status, and astroglial reaction.

BACKGROUND: The totally gastrectomized (TGX) rat is a new experimental model with which to produce widespread spongy vacuolation in spinal cord (SC) white matter, strongly reminiscent of that observed in subacute combined degeneration (SCD) of human SC. EXPERIMENTAL DESIGN: We did in long-term experiments combined biochemical and histologic studies on SCs from both TGX-rats and rats fed a cobalamin-deficient (Cbl-D) diet. We also investigated the effects of single in vivo administration of some neurotrophic growth factors on the activity of L-ornithine decarboxylase (ODC) (the key-point in the polyamine biosynthetic pathway) in rat SC. RESULTS: Biochemically, ODC activity was still induced 3 and 6 months after total gastrectomy (TG), while it did not change significantly even after 9 months of feeding a Cbl-D diet. Both TG and feeding the Cbl-D diet greatly decreased the cobalamin level in both serum and SC, although these decreases occurred more slowly in rats fed a Cbl-D diet. Nerve growth factor did not induce ODC in either Cbl-D myeloneuropathy; epidermal growth factor induced ODC in both Cbl-D myeloneuropathies. Basic fibroblast growth factor induced SC ODC only in TGX-rats. Histologically, spongy vacuolation was still widespread 3 and 6 months after TG, while it was spotty even after 9 months of feeding a Cbl-D diet. There was massively increased staining of astrocytes positive for glial fibrillary acidic protein, mainly in the gray matter, in both Cbl-D myeloneuropathies. Finally, repeated in vivo injections of cobalamin to TGX rats only partially reduced ODC induction, the severity of spongy vacuolation, and the increase in glial fibrillary acidic protein-positive astrocytes. CONCLUSIONS: These results suggest: (a) ODC induction is a persistent and inherent feature in the TG-induced SCD of rat SC; (b) an increase in glial fibrillary acidic protein positive astrocytes in rat SC is not mandatorily connected with an increase in polyamine biosynthesis; (c) a mere deficiency of Cbl seems to be not the only key-point in the pathogenesis of the ODC induction and of the SCD-like lesions, both brought about in rat SC by TG.

Subacute combined degeneration and induction of ornithine decarboxylase in spinal cords of totally gastrectomized rats.

Totally gastrectomized rats have been used to induce a spongy demyelination in the white matter of the spinal cord (SC) which is strongly reminiscent of that observed in subacute combined degeneration of human SC. Totally gastrectomized rats are deprived of intrinsic factor and thereafter become deficient in cobalamin. Morphologically, the spongy demyelination of the white matter of the rat SC, was evident 2 months after total gastrectomy. Biochemically, we investigated the hypothesis that polyamine biosynthesis might be deranged in the rat SC with experimental subacute combined degeneration, since polyamines are well known to be bound to myelin in the mammalian central nervous system. We measured the levels of both the polyamine biosynthetic decarboxylases, L-ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase, the key points in the polyamine biosynthetic pathway, in these SC. There was a sharp increase in ODC activity in SC 2 months after total gastrectomy, without significant changes in S-adenosyl-L-methionine decarboxylase activity. The increase in ODC activity seems to be organ-specific and was not due to a proliferation of neuroglial cells. Interestingly enough, the same morphologic and biochemical features found in SC of 2-month-totally-gastrectomized rats were present also in SC of newborn rats, which indeed showed incomplete myelination, vacuolated appearance, and an ODC activity level higher than that of adult SC. Therefore, total gastrectomy seems to induce a type of regression in the SC of totally gastrectomized rats toward neonatal life, at least in terms of the degree of myelination and of ODC activity level. Biochemically, no changes in ODC activity were observed in SC of rats fed a cobalamin-deficient diet for 3 months. Morphologically, only a proliferation of neuroglial cells with a moderate demyelination was observed in SC of these rats maintained on a cobalamin-deficient diet for 3 months.

Polyamine biosynthetic decarboxylases in muscles of rats with different experimental myopathies.

The activities of the two polyamine biosynthetic decarboxylases (PBD), L-ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAMD), have been measured in quadriceps femoris of rats killed at different times after the induction of calciphylaxis- or serotonin(5-HT)-induced myopathy. Decreases in both PBD levels were observed at early times after both myotoxic treatments. Subsequent progressive increases in both enzyme levels were observed to nearly control values by 4 days after 5-HT administration. In the 5-HT-treated rats, the effects on the myocardial PBD activities were different from those in skeletal muscle, with no effect on ODC but much on SAMD, when rats were killed shortly after 5-HT injection. These results demonstrate that the time-course of the changes in PBD activities in quadriceps femoris mirrors quite well the successive occurrence of degenerative and regenerative processes during the calciphylaxis-induced myopathy and the 5-HT-induced myopathy; it is 5-HT that is mainly responsible for the decreases in PBD levels observed in both experimental myopathies, since dihydrotachysterol alone was without any effect on PBD activity levels and 5-HT alone was effective; myocardial ODC reacts more slowly to 5-HT than quadriceps femoris ODC.