Discussion

Table 1 shows that metalloporphyrins can form single strand breaks or alkali labile sites upon light irradiation as can porphyrins without chelated metal atoms (11,12). Gomer et al. (12) concluded that this type of DNA-damage did not increase the number of mutations in Chinese hamster cells. It remains to be seen whether metalloporphyrin photosensitisation may be mutagenic. Before clear evidence is obtained, one should not completely exclude the possibility, although it is most likely that metalloporphyrins photosensitise the cells by the same mechanisms as other porphyrins. The basic mechanism is probably by production of singlet oxygen (20). Whether or not singlet oxygen is mutagenic, is debated, but recent findings indicate that pure singlet oxygen can induce mutations in a virus based vector (21).

Data on photooxidation of tryptophan (Table II) and the data of others (5,22) indicate that SnPP and ZnPP are stronger photosensitizers than HP and PP and indeed stronger than the Cr-derivatives. However, the relative efficiency is strongly dependent on the wavelength of the phototherapy light as well as the system used to assay photosensitisation. The absorption spectra of metalloporphyrins are relatively similar but different from the absorption spectra of HP and PP. The differences in the spectral region around 450 nm is of great importance for phototherapy, since blue light is widely used. The Soret band of the metalloporphyrins are red shifted by 10 to 30 nm compared to the Soret bands of HP and PP which are close to 400 nm (data not shown). It may be assumed that the differences in photosensitising efficiency between SnPP/ZnPP and HP/PP partly can be explained by differences in absorption spectra.

In methanol, the photosensitising efficiency of SnPP and ZnPP seem to be relatively similar and stronger than in aqueous solution (data not shown). McDonagh and Palma (3) reported that the photodegradation of bilirubin under white light was enhanced 12-fold by 1,5 mM SnPP which corresponds to our data. Furthermore, it has been shown that the sensitising efficiency was lower in the presence of albumin.

Scott et al. (22) compared different porphyrins and metalloporphyrins with respect to photosensitisation under long wavelength light. Under their conditions ZnPP did not cause hemolysis of red blood cells, while SnPP did. On the other hand, both metalloporphyrins may cause enzyme inactivation (22). One important factor determining the localisation and types of cellular effects is probably the uptake of the porphyrins. In the TMG-1 cells, ZnPP was taken up much more efficiently and caused more DNA-damage (Fig. 1, Tab. I). Differences in the relative amounts of membrane damage and DNA-damage have been shown to be strongly dependent on the binding sites for porphyrins in human cells (23,24). As indicated in Table I, ZnPP is a stronger photosensitizer of DNA than SnPP, while the cell killing efficiency is relatively similar (Fig. 2). The different localisation of the two metalloporphyrins may explain their different behaviour.

It is likely that the metalloporphyrins may be reached by light while present in human tissue. Photo-induced killing of newborn rats has been observed after injection of metalloporphyrins (25,26).

Light exposure of metalloporphyrins used as chemotherapeutic compounds in newborns will induce photochemical reactions, not solely dependent on the photosensitising properties of the porphyrins, but also on the properties of bilirubin present in the tissues of hyperbilirubinemic newborns. It has been shown that bilirubin may induce photosensitising effects (6,7,8,9). Bilirubin has also been reported to be a scavenger of peroxyl radicals generated chemically (13,14,15) and to reduce the production of superoxide by polymorphonuclear leukocytes (27). Part of the latter effect may be of cytotoxic nature, but it is probable that bilirubin is a natural antioxidant. Our hypothesis was that bilirubin could reduce the cytotoxity by scavenging singlet oxygen produced by porphyrin photosensitisation.

The data on survival of mouse cells after photosensitisation by metalloporphyrins in the presence and absence of bilirubin (Fig. 2) were analysed further by doing some theorethical considerations. When different agents are combined, they may act independently or interact by synergism or antagonism. If there are no interactions, one may expect that the surviving fractions after a certain light dose are related in the following way:

(S_bilirubin ^. S_Porphyrin)/S_{bilirubin+porphyrin}= 1

where the subscripts indicate the agent(s) present during irradiation. The hypothesis that there are no interactions between the phototoxic action of porphyrins and bilirubin was tested by calculating the ratios between the survival values for 10 - 11 separate observations for SnPP and ZnPP, respectively(mean + S.E.):

(S_bilirubin ^. S_SnPP)/S_{bilirubin+SnPP}= 1.07 + 0.08

(S_bilirubin ^. S_ZnPP)/S_{bilirubin+ZnPP}= 0.75 + 0.15

None of the means were significantly different from 1, and this indicates that bilirubin does not interact with the photodynamic inactivation of cells by metalloporphyrins.

[Up]