Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

Summary The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E₂) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E₂ via the C2- and C16-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E₂ metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 hydroxylation of E₂. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16 hydroxylation of E₂, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E₂ metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E₁), 0.69 µM 2-hydroxyestrone (2-OHE₁), and 0.66 µM 2-methoxyestrone (2-MeOHE₁) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E₂ metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 hydroxylation in favor of C16-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E₂ metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HPR N-(4-hydroxyphenyl) retinamide - I3C indole-3-carbinol - TAM tamoxifen - E₂ 17-estradiol - E₁ estrone - 2-OHE₁ 2-hydroxyestrone - 2-MeOHE₁ 2-methoxyestrone - 16-OHE₁ 16-hydroxyestrone - E₃ estriol - DME/F12 Dulbecco's modified Eagle's medium - F12 Ham's medium - HU hydroxyurea - PBS phosphate buffered saline - NaOH sodium hydroxide - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - [C2-³H] E₂ estradiol labeled at C2 position - [C16-³H] E₂ estradiol labeled at C16 position - ANOVA analysis of variance     

The influence of stimulus parameters on the visual field indices by automated projection perimetry

The various stimulus parameters offered by two standard automated projection perimeters [Humphrey Field Analyser 630 (HFA) and Octopus 2011, namely, stimulus size and location and the interaction of adaptation level and stimulus duration, were compared in a sample of 20 patients attending a glaucoma clinic using the visual field indices mean defect (MD), loss variance (LV), short-term fluctuation (SF) and corrected loss variance (CLV). LV and SF were greater with Octopus program 32 compared with Octopus program G1 (P < 0.02). No difference in the indices was found between stimulus sizes I and III for HFA program 30-2. MD was greater for program 30-2 compared with program 32 (P < 0.002) when expressed in terms of log (L/L) whereas LV (P < 0.02) and SF (P < 0.02) were greater for program 32. All differences were considered to be negligible in the clinical sense.     

Previous abdominal colectomy affects functional results after ileal pouch-anal anastomosis

We assessed the effect of previous abdominal colectomy on functional results after ileal J pouch-anal anastomosis (IPAA) in patients with ulcerative colitis. Twenty-five patients with colectomy prior to IPAA were compared with 22 patients who underwent noncolonic abdominal operations prior to IPAA. No differences were observed in pre- or postoperative resting anal sphincter pressure, squeeze pressure, or rectal inhibitory reflex. Previous colectomy was associated with a greater incidence of postoperative small bowel obstruction. Mean ± SEM daily stool frequency at 1 and 12 months postoperatively, respectively, was 8.9±0.8 and 5.7±0.3 for patients who had undergone previous colectomy, and 8.2±0.7 and 6.0±0.5 for the no previous colectomy group (p=not significant). At the same postoperative intervals, nocturnal stool frequency was 1.9±0.3 and 1.1±0.2 for the colectomy group and 1.5±0.3 and 0.6±0.1 for the no colectomy group (p=0.05 at 1 year). More patients in the previous colectomy group had greater than or equal to 1 nocturnal stool after 1 year (71% versus 33%,p=0.03). Although pouch capacity at 1 year was not different in the 2 groups, pouch capacity was directly related to stool frequency in the no colectomy group (r²=0.48,p=0.01), but not in the previous colectomy group (r²= 0.08,p=not significant). We conclude that previous abdominal colectomy may be associated with a higher overall incidence of small bowel obstruction. Moreover, previous colectomy is a determinant of postoperative nocturnal stool frequency after IPAA, most likely due to altered ileal pouch function. When possible, single-stage colectomy, mucosal proctectomy, and endorectal ileal pouch-anal anastomosis should be performed in patients requiring colectomy for ulcerative colitis.

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Resumen Hemos valorado el efecto de una colectomía abdominal previa sobre los resultados funcionales después de anastomosis ileoanal de bolsa en J (AIAB) en pacientes con colitis ulcerativa. Veinticinco pacientes con colectomía previa a la AIAB fueron comparados con 22 pacientes sometidos a operaciones abdominales no colónicas antes de la AIAB. No se hallaron diferencias en cuanto a la presión en reposo del esfínter anal (preoperatoria o postoperatoria), a la presión de compresión, o al reflejo rectal inhibitorio. La colectomía previa apareció asociada con una mayor incidencia de obstrucción del intestino delgado. La frecuencia de defecación diaria a 1 y a 12 meses postoperatorios, respectivamente, fue 8.9±0.8 y 5.7±0.3 para los pacientes que habían sido sometidos a colectomía previa, y 8.2 ±0.7 y 6.0±0.5 para el grupo sin colectomía previa (p=NS). En los mismos períodos postoperatorios la frecuencia de defecación nocturna fue 1.9±0.3 y 1.1±0.2 para el grupo con colectomía previa y 1.5±0.3 y 0.6 ±0.1 para el grupo sin colectomía (p=0.05 a 1 año). Más pacientes en el grupo de colectomía previa presentó más de una o una deposición nocturna después de un año (71% versus 33%, p=0.03). Aunque la capacidad de la boisa a un ano no apareció diferente en los 2 grupos, la capacidad de la bolsa apareció directamente relacionada con la frecuencia de la deposición en el grupo sin colectomía (r²=0.48,p=0.01), pero no en el grupo con colectomía previa (r²=0.08,p=NS). Nuestra conclusión es que una colectomía abdominal previa puede estar asociada con una mayor incidencia de obstrucción del intestino delgado. Además, la colectomía previa aparece como un factor determinante de la frecuencia de defecación nocturna después de AIAB, muy probablemente por alteración de la función de la bolsa ileal. En cuanto sea posible, se debe realizar la colectomía, proctectomía mucosal, y anastomosis ileal endorrectal en una sola etapa en los pacientes que requieran colectomía por colitis ulcerativa.

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Résumé Nous avons chercher à savoir si le fait d'avoir déjà effectué une colectomie retentissait sur les résultats de fonctionnement de l'anastomose iléo-anale avec réservoir en J (AIAR) chez le patient avec rectocolite hémorragique. Vingt cinq patients ayant eu une colectomie avant d'être opérés de leur AIAR ont été comparés à 22 patients ayant une intervention abdominale sans colectomie avant d'être opérés de leur AIAR. Aucune différence dans la pression sphinctérienne au repos pré ou post-opératoire, dans la pression de contraction ou dans le réflexe inhibiteur rectal n'a été observée. La colectomie préalable était associée à une incidence élevée d'occlusion intestinale post-opératoire. Le nombre de selles à 1 et à 12 mois post-opératoires était de 8.9±0.8 et 5.7±0.3, respectivement, chez le patient sans chirurgie colique antérieure (NS). Aux mêmes intervalles, la fréquence de selles nocturnes était de 1.9 ±0.3 et de 1.1±0.2 pour le groupe à colectomie préalable et de 1.5±0.3 et 0.6±0.1 pour le groupe sans chirurgie colique préalable (p=0.05 à un an). Dans le groupe à colectomie préalable, il y avait plus de patients qui avaient une ou plusieurs selles nocturnes après la première année (71% versus 33%;p= 0.03). Bien que la capacité du réservoir ne différait pas à 1 an entre les 2 groupes, la capacité était directement en rapport avec la fréquence des selles dans le groupe sans chirurgie colique préalable (r²=0.48; p=0.01), mais sans rapport dans le groupe avec chirurgie colique préalable (r²=0.08, p=NS). Nous concluons que la colectomie préalable est asscoiée à une incidence d'occlusion post-opératoire supérieure. De plus, la colectomie préalable est associée à une fréquence plus élevée de selles nocturnes après AIAR, probablement liée à un dysfonctionnement du réservoir. Lorsque la colectomie totale avec mucosectomie rectale, avec anastomose iléo-anale et réservoir est indiquée chez le patient ayant une rectocolite hémorragique, il vaut mieux la faire en un seul temps.

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Presented at the Société Internationale de Chirurgie, Toronto, Ontario, Canada, September, 1989.     

Variability of the steady-state visually evoked potential: Interindividual variance and intraindividual reproducibility of spatial frequency tuning

At low contrast levels there is good agreement between the psychophysical contrast sensitivity function and the tuning curve of the visually evoked potential (i.e., VEP amplitude vs spatial frequency). At high contrast, however, some researchers have found bimodal VEP tuning curves whereas others have not. We studied the VEP in 22 subjects in a short-term cross-sectional study and in 13 subjects in a longitudinal study over 8 sessions covering 28 days. Grating stimuli with 60% contrast were square-wave modulated in time (7.8 reversals/s) and space (0.06–16 cycles/degree). We found large interindividual variance in the shape of the tuning curves; about half of the subjects showed a unimodal shape, while the other half showed a bimodal one (with a notch between 1 and 2 cycles/degree). These features turned out to be stable in the longitudinal study, where variability could mainly be ascribed to a multiplicative influence common to all spatial frequencies. The marked interindividual differences in the shape of the tuning curve, which seem to be intraindividually stable, may explain previous discrepancies. It is not yet clear why the notch exists in about half of our subjects.     

Proliferative vitreoretinopathy — is it anything more than wound healing at the wrong place?

Proliferative vitreoretinopathy (PVR) is a reactive process of the ocular tissue after perforating trauma, retinal detachment, and surgical manipulations. Although several studies, most of them experimental, have focused on the detection of specific etiologic factors in the development of PVR, there is compelling evidence that PVR is nothing more than a physiologic tissue repair process with undesirable consequences for the retina. Important features of PVR involving the role of platelets, mononuclear phagocytes, and fibroblasts parallel the chain of events observed in tissue repair elsewhere in the body. Numerous experimental models for PVR, originally designed to find specific stimuli for the generation of intraocular traction membrane formation, have shown that the process of PVR is the common pathway of the eye's reaction to vitreoretinal trauma of any kind. Accordingly, vitreoretinal surgeons could learn a lot from the work of other disciplines, e.g. surgery and dermatology, on wound healing, and the factors known to modify wound healing elsewhere in the body should be taken into consideration. The well-established impairment of tissue repair processes caused by medical treatment with corticosteroids and cytotoxic agents suggests a combined medical approach to PVR as an adjunct to surgical treatment, using refined methods of application and dosage. Steroids and cytotoxic drugs will influence the course of PVR by suppressing macrophage recruitment and the initial inflammatory reaction as well as the proliferative phase of wound healing with traction retinal detachment, respectively.     

Study of intact (1–84) parathyroid hormone secretion in patients undergoing parathyroidectomy

Patterns of intact (1–84) parathyroid hormone (intact PTH) elimination and subsequent recovery of parathyroid function were studied in 12 patients undergoing parathyroidectomy. Nine patients had primary hyperparathyroidism (HPT), with single gland disease in 6 and multiple gland disease in 3. Two patients had subtotal parathyroidectomy for HPT secondary to chronic renal failure and 1 underwent excision of a hyperfunctioning parathyroid autograft. Using a sensitive 2-site immunochemiluminometric assay, serum intact PTH levels were measured preoperatively, intraoperatively, and postoperatively. A dual phase pattern of hormone clearance was found in 10 of the 12 patients, including the patient undergoing autograft excision. A monoexponential clearance pattern was seen in the remaining 2 patients, both of whom had subtotal parathyroidectomies for multiple gland disease. In the patients with primary HPT due to single gland disease, the early phase of intact PTH clearance had a half-life (T1/2) of 3.3 (±standard deviation 0.9) minutes and a late T1/2 of 96.4 (±standard deviation 92.7) minutes. Calculation of decay curves and half-lives for the patients undergoing subtotal parathyroidectomy was more difficult because of the inherent uncertainty in determining time zero. Nevertheless, in all but 2 patients, the clearance pattern was biexponential and the T1/2 measurements were very similar to those encountered in patients with single-gland disease. In the 2 patients with monoexponential clearance, the T1/2 figures were 86.7 minutes and 26.7 minutes, respectively. In the patients undergoing parathyroidectomy for primary HPT, levels of intact PTH were lowest at 1–3 hours after surgery, recovering to normal in the majority of patients by 18–40 hours.

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Resumen Los patrones de elminación de la hormona paratiroidea intacta (1–84) y la subsiguiente recuperación de la función paratiroidea fueron estudiados en 12 pacientes sometidos a paratiroidectomía. Nueve presentaban hiperparatiroidismo (HPT) primario, con enfermedad de una sola glándula en 6 y de múltiples glándulas en 3. Dos fueron sometidos a paratiroidectomía subtotal por HPT secundario o falla renal y uno a resección de un autoinjerto de paratiroides hiperfuncionante. Mediante una sensible determinación inmunoquimioluminométrica, se midieron los niveles séricos de PTH intacta en las fases pre-, intra-, y postoperatorias. Se encontró un patrón bimodal de depuración (clearance) hormonal en 10 de los 12 pacientes, incluyendo el caso de la resección del autoinjerto hiperfuncionante. En los otros 2 pacientes se observó un patrón monoexponencial de depuración; ambos fueron sometidos a paratiroidectomía subtotal por enfermedad glandular múltiple. En los pacientes con HPT primario por enfermedad glandular única, la fase temprana de dupuración de la PTH intacta tuvo una vida media de 3.3 (±0.9 DE) minutos y la fase tardía una vida media de 96.4 (±92.7 DE) minutos. Los cálculos de las curvas de descenso y las vidas medias en los pacientes sometidos a paratiroidectomia subtotal fueron más complicados debido a dificultad inherente para determinar la hora cero. Sin embargo, en todos los pacientes, menos en 2, el patrón de depuración fue bioexponencial y las vidas medias fueron muy similares a los encontrados en pacientes con enfermedad de una sóla glándula. En los 2 pacientes con depuración monoexponencial, las cifras de vida media fueron 86.7 minutos y 26.7 minutos, respectivamente. En los pacientes sometidos a paratiroidectomía por HPT primario, los niveles de PTH intactos llegaron a sus más bajos niveles las 1–3 horas después de la cirugía, con recuperación a valores normales a las 18–40 horas en la mayoría de los pacientes.

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Résumé L'élimination d'hormone parathyroïde entière (1–84) (PTH intacte) et la récupération postopératoire de la fonction parathyroïdienne ont été étudiées chez 12 patients ayant eu une parathyroïdectomie. Neuf patients avaient une hyperparathyroïdie (HT) primaire (HTI); 6 avaient une seule glande atteinte; 3 avaient une atteinte multiglandulaire. Deux patients ont eu une parathyroïdectomie subtotale pour HT secondaire à une insuffisance rénale chronique et un patient a dû être opéré pour enlever une autogreffe parathyroïdienne hyperfonctionnelle. Grâce à l'analyse immunochémoluminométrique à 2 sites, sensible, les taux de PTH intacte ont été mesurés en pré-, per-, et postopératoire. Une phase double de clairance hormonale a été retrouvée chez 10 des 12 patients, y compris le patient ayant eu une excision de greffe. La courbe d'élimination de la PTH avait une allure monoexponentielle chez les 2 autres patients, tous 2 ayant eu une parathyroïdectomie subtotale pour maladie multiglandulaire. Chez les patients ayant une HTI en rapport avec une seule parathyroïde, la phase précoce de la clairance de PTH intacte correspondait à une demi-vie (T1/2) de 3.3 (± DS 0.9) minutes alors que la T 1/2 tardive était de 96.4 (± DS 92.7) minutes. Le calcul des courbes de décroissance et de la T1/2 des patients ayant une parathyroïdectomie subtotale était plus difficile étant donné l'impossibilité de déterminer avec précision le temps zéro. Néanmoins, chez tous les patients sauf 2, la courbe d'élimination était d'allure biexponentielle et les mesures de la T1/2 étaient très semblables à celles des patients ayant une seule parathyroïde malade. Chez les 2 patients ayant une clairance d'allure monoexponentielle, la T1/2 était respectivement de 86.7 et 26.7 minutes. Chez les patients ayant eu une parathyroïdectomie pour HT, les taux de PTH intacte étaient au plus bas 1 à 3 heures après l'intervention et remontaient à la normale chez la plupart des patients entre 18 et 40 heures.

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Presented at the International Association of Endocrine Surgeons in Toronto, Ontario, Canada, September, 1989.     

Clinical toxicity associated with tiazofurin

Summary Tiazofurin, an investigational antimetabolite, is undergoing clinical evaluation in leukemia. We analyzed the data base of 198 patients entered in Phase I trials to characterize the incidence and severity of toxicities associated with tiazofurin according to dose and schedule. Severe myelosuppression occurred infrequently, and was not dose-dependent. A five day bolus schedule had a higher incidence of severe or life-threatening neutropenia than other schedules. Tiazofurin produced lymphopenia which was not dose-dependent in the range of 23–36% decrease from baseline, and the effect on lymphocyte count was generally greater than the decline in neutrophil count. Non-hematologic toxicity of a moderate or worse severity ( grade 2) included nausea and vomiting (18% of all courses), serum transaminase elevations (SGOT, 16%; SGPT, 9%), rash (9%), stomatitis (3%), conjunctivitis (3%), headache (10%), other signs of central nervous system toxicity (8%), and cardiac toxicity, primarily pleuropericarditis (4%). Dose-related cutaneous toxicity, headache, and nausea and vomiting were evident in the five day bolus schedule, and myalgia was more frequently reported at higher doses on the single dose schedule. The five day continuous infusion (CI) schedule had a higher incidence of neurotoxicity, cardiac toxicity, SGPT elevations and ocular toxicity than the daily for five days bolus schedule, but none of these differences attained statistical significance. Although the peak plasma concentrations of tiazofurin achieved with the five day bolus schedule were 3-fold higher than the steady-state plasma levels seen with an equal dose given by CI, the area under the concentration-time curve (AUC) was approximately 1.6-fold higher with CI. These observations suggest that both high peak plasma concentrations (above 400 uM) and prolonged exposure to plasma levels exceeding 50 uM may result in a higher incidence of serious non-hematologic toxicity.     

Early phase II Gynecologic Oncology Group experience with ifosfamide/mesna in gynecologic malignancies

Starting in July 1985, the Gynecologic Oncology Group conducted a series of phase II trials with ifosfamide/mesna in advanced or recurrent gynecologic malignancies. Previously untreated patients received 1.5 g/m² i.v. ifosfamide daily for 5 days. Mesna was given i.v. q4h×3 following ifosfamide; each dose was 20% of the daily ifosfamide dose. All patients with ovarian and 87% of those with cervical cancer had previously undergone platinum-based therapy. Because of the toxicity encountered in previously treated patients with ovarian carcinoma, the dose of ifosfamide was reduced to 1.2 g/m² daily in all patients who had received prior chemo- or radiotherapy. In epithelial ovarian carcinoma, responses were observed in 8 (20.0%) of 41 evaluable patiens, with 3 (7.0%) complete responses. Response duration was 2.1–20.3+ months, with a median of 6.9+ months. In squamous-cell carcinoma of the cervix, 3 (11.1%) of 27 evaluable patients showed partial responses of 1.8, 2.2, and 3.1 months' duration. Of 26 untreated patients with mixed mesodermal tumors of the uterus, 5 (19.2%) achieved complete and 3 (11.5%) showed partial responses, for an overall response rate of 30.7%. Response duration was 1.4+-8.6 months, with a median of 3.8 months. Toxicity included two deaths due to renal insufficiency and a third related to neurologic impairment. Hematologic toxicity was manageable. Ifosfamide/mesna has activity in a wide range of gynecologic malignancies.Presented at the Satellite Symposium Ifosfamide in Gynecological Tumors of the 5th European Conference on Clinical Oncology and Cancer Nursing, London, September 3–7, 1989     

Comparative pharmacokinetics of escalating doses of doxorubicin in patients with metastatic breast cancer

Summary Recombinant human granulocyte colony-stimulating factor (G-CSF) has been shown to reduce neutropenia following cytotoxic therapy, thereby enabling dose escalation to improve the response rate. It is important to know whether drug kinetics change as doses are increased. Doxorubicin was selected because of its broad spectrum of activity and its known efficacy in metastatic breast cancer. Doses of 75, 100, 125 and 150 mg/m² were given to 11 patients with metastatic breast cancer by infusion over 30 min. Serum concentrations of parent drug and metabolites were determined during the first 48 h following the infusion by high-performance liquid chromatography (HPLC). The serum concentration vs time curve decayed as a triple exponential function in four patients and as a double exponential function in seven. A four-compartment model, one central and three peripheral, would predict concentrations to within 1 SE of the observed values. Doxorubicinol was the principal metabolite, and doxorubicinone and 7-deoxydoxorubicinone were clearly identified. There was a linear increase in the AUC with dose. In addition, a small and transient increase in circulating levels of doxorubicinol and other important metabolites was observed 6 h following the administration of doxorubicin, which suggests the existence of an enterohepatic, or other, re-circulation mechanism. We conclude that in the dose range selected the kinetics of doxorubicin are linear and that the increase in toxicities seen with the higher doses of doxorubicin, following the second and third fortnightly administration, may be due to intracellular drug accumulation in tissues.