Correlación de Clima Laboral y Estilos de Liderazgo considerando las

tissue responds differently to neoplastic tissue. A similar level o f increased oxygen tension was measured with 5 and 30 minutes carbogen prebreathing indicating no PIBT dependence. Furthermore, the same response has also been seen in the subcutis o f C3H mice bearing RIF-1 tumours (D. Honess, personal communication). Normal tissue vasodilation during carbogen breathing would explain a sustained improvement in subcutaneous p02 However, improved normal tissue blood flow could also result in a steal-effect from the tumour, decreasing it’s supply o f oxygen. Hence one could speculate that during the first few minutes o f carbogen breathing, tumour p0 2 appears to

increase until the vascular response, mediated by hypercapnia and hyperoxia, take effect. Vasodilation o f normal tissue could result in an oxygen steal from the tumour, reducing the efficacy o f carbogen breathing in the tumour tissue but not in the normal tissue.

However, this does not explain the second transient increase in SaF pOz seen with a prebreathing time o f 45 minutes. This theory is based upon the assumption that the histograph pOz measurements are representative, and the sampling size is adequate. Although, in support o f the Eppendorf histograph, is the fact that the results were reproducible, and that the carbogen prebreathing dependencies shown in figure 5.1 at 30, 45, and 60 minutes were, in each case, the pooled results from two separate groups o f animals from different passages.

Interestingly, real-time pOi measurements with the LICOX CMP and the fibre-optic sensor did not reveal any PIBT dependence consistent with the Eppendorf histograph data during carbogen breathing in the sarcoma F. Instead, both sensors showed an increase in oxygenation which was maintained for the duration o f the breathing episode. In chapter 3 the LICOX electrode was shown to be susceptible to contamination fi'om atmospheric and subcutaneous sources o f oxygen. Although larger tumours were used in these studies, and bearing in mind that the Eppendorf data were very reproducible, skin contamination is probably the most likely reason for the discrepancies between the two electrodes. Such contamination could explain, (a) a higher pO: reading during carbogen breathing compared to those measured with the fibre-optic sensor or the Eppendorf pOi histograph, and (b) no PIBT dependence. However, contamination can not explain the lack o f PIBT dependence measured with the fibre-optic sensor. The sample size for the fibre-optic sensor was a lot smaller than that o f the histograph. For each time point, at least 5 tumours were measured during the histograph studies, and with between 50-100 individual pOi measurements in each tumour, the sample size was therefore o f the order o f several hundred measurements. In contrast, the fibre-optic studies were performed at a single insertion site in 20 tumours, thus large sampling differences between the two investigations exist. However, the tight error bars for the fibre-optic data {cf. figure 5.6) possibly argue against this conclusion.

In addition to measuring changes in oxygen tension, this chapter has also shown that radiosensitisation can be improved in subcutaneous sarcoma F tumours by breathing carbogen. A comparison o f the radiosensitisation data with the oxygenation status, as judged by the percentage o f values less than 2.5 mmHg, shows a qualitative, but no

radiation response with 5 and 45 minutes carbogen prebreathing, but in reality no PIBT dependence was seen in the radio sensitisation studies. A lack o f PIBT dependence for tumour irradiation response during host carbogen breathing is in contrast with other tumour models (Inch et al, 1970; Suit et al, 1972; Siemann et al, 1977). It is possible that the absence o f a direct quantitative correlation between these measurements may reflect the fact that radiosensitisation is determined by the oxygenation status o f the cells yielded from a digest in a clonogenic assay, whereas pOz values are obtained from a finite number o f regions in each tumour. Thus the sample size for a clonogenic assay is probably 1 or 2 magnitudes larger than for the pOz histography. In addition, these discrepancies could reflect the fact that the measurements were performed on different groups o f tumours, although this is less likely, since the results presented here have been reproduced, and mirror those reported by others (Hill et al, 1997, submitted)', indicating very little inter-passage variation in the sarcoma F. One final possibility can be derived fi’om the speculation regarding nitric oxide in chapter four. If the concentration o f nitric oxide had been increased by carbogen to a high enough level to cause direct DNA damage fixation, then measurements o f oxygen tension may not necessarily reflect surviving fi-action.

Other studies in this chapter have also shown the response o f tumour pOz to carbogen breathing when in combination with two modifiers o f perfusion-limited hypoxia. In combination with nicotinamide, the pOz-PIBT dependence was reduced in the SaF. Furthermore, the suboptimal effect o f breathing carbogen for 60 minutes was reversed in the SaS and HT-29, causing increases in pOz following the addition o f nicotinamide; this is also suggestive o f an elimination o f a PIBT dependence in these two tumours. This in agreement with the studies o f Chaplin et a l (1993) who showed that nicotinamide could reduce the critical importance o f carbogen PIBT in SCCVII murine tumours. Improvements in tumour oxygenation should be related to nicotinamide’s ability to normalise tumour blood flow (Chaplin et al, 1990; Hill and Chaplin, 1995) facilitated at least in part by inhibiting leukocyte adhesion (Hiromatsu et al, 1991) and increasing leukocyte filterability (Honess et al, 1996). This would prevent transient blood vessel blockage leading to acute hypoxia. Normalisation o f tumour blood flow alone would probably lead to changes in oxygenation beyond the resolution o f pOz histography, and indeed pOz measurements made following administration o f nicotinamide on its own in

all four tumours showed no significant difference to untreated controls. Nicotinamide may also inhibit transcription o f the nitric oxide synthase gene, and prevent translation o f existing mRNA transcripts (Pellat-Deceunynck et al, 1994). This could reduce normal tissue vasodilation, improving blood flow and oxygen delivery to the tumour.

The combination o f pentoxifylline and carbogen breathing has been reported to improve tumour oxygenation and perfiision (Teicher et al, 1993; Honess et al, 1993, 1995a, 1996; Lee et al, 1993, 1994; Song et al, 1992). Studies have shown that pentoxifylline can act as a vasodilator (Kaapa et al, 1991; Kaye et al, 1996), increase red cell deformability, reduce blood viscosity, decrease platelet aggregation and thrombus formation (Ward and Clissold, 1987), and increase leucocyte filterability (Honess et al, 1996). The studies presented in this chapter are in agreement with these observations: SaF p02 was shown to increase following 30 minutes carbogen breathing in combination with pentoxifylline. However, this was the only combination o f the two agents to achieve a therapeutic improvement in oxygenation (indicated by a decrease in the fraction o f radiobiological hypoxia), and interestingly, a dose o f pentoxifylline reduced the effectiveness o f 5 minutes carbogen breathing alone. These results are both surprising and difficult to explain, since one might have expected a sustained increase in tumour p0 2 similar to that observed with nicotinamide and carbogen breathing.

5.4 Summary

This chapter has shown significant improvements in oxygenation in the sarcomas F and S, and the human tumour xenograft, HT-29 are achievable by host carbogen breathing. p02 levels in the carcinoma NT, however, did not improve during carbogen inhalation. The increases in p02 were time dependent supporting the existence o f a pre-irradiation breathing time dependence, although, radiosensitisation with carbogen breathing in the sarcoma F had no PIBT dependency.

A carbogen prebreathing dependency was not seen in p02 measurements in the SaF made with the LICOX electrode or the fibre-optic sensor, although p02 values did significantly improve. However, practical and/or technical limitations with these sensors may have

Eppendorf pOz histograph. Combining carbogen breathing with nicotinamide eliminated the PIBT dependence in the SaF, causing increased pOz with previously suboptimal levels o f carbogen administration. A similar result was seen with 60 minutes o f carbogen breathing in the SaS and HT-29. In combination with pentoxifylline, a carbogen PIBT o f 30 minutes caused a reduction in the fraction o f readings less than 2.5 mmHg in the SaF.

The influence o f the carbon dioxide content in carbogen-type gases was also investigated using the sarcoma F. With a PIBT o f 5 minutes, reducing the CO2 content from 5% to

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Carbogen flow rate (litres/min)

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Prebreathing Time (minutes)

Figure 5.1: Effect o f carbogen breathing on the sarcoma F. Upper panel: Dependence o f tumour p 0 2 on carbogen flow rate at a fix ed prebreathing time o f 5 minutes. Lower panel: Dependence o f tumour p 0 2 on length o f carbogen breathing at a fixed flo w rate

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