Selective accumulation of endogenously produced porphyrins in a liver metastasis model in rats

Summary As an initial approach to optimize 8-aminolaevulinic acid (6-ALA)-induced photosensitization of tumours, we examined the response of three enzymes of the haem biosynthetic pathway: 8-ALA dehydratase, porphobilinogen deaminase (PBGD) and ferrochelatase. Only PBGD activity displayed a time-and dose-related increase in tumours after intravenous administration of 300 mg kg-1 6-ALA. The time course for porphyrin fluorescence changes, reflecting increased production of the penultimate porphyrin, protoporphyrin IX (PPIX), showed a similar pattern to PBGD. This apparent correlation between PBGD activity and porphyrin fluorescence was also observed in four cultured tumour cell lines exposed to 0.1-2.0 mm 6-ALA in vitro. The increase in PBGD activity and PPIX fluorescence was prevented by the protein synthesis inhibitor cycloheximide. As the apparent Km for PBGD was similar before and after 6-ALA, the increase in PBGD activity was attributed to induction of enzyme de novo. These observations of an associated response of PBGD and PPIX imply that PBGD may be a ratelimiting determinant for the efficacy of 6-ALA-induced photosensitization when used in photodynamic therapy.Keywords: 6-aminolaevulinic acid: photosensitization; porphobilinogen deaminase: haem biosynthesis; porphyrin fluorescence Haem is an essential prosthetic group in many critical cellular proteins such as haemoglobin, cytochrome P450 and cyclooxygenase (Abraham, 1991). Eight enzymes are involved in the biosynthesis of haem, a process that occurs in two subcellular compartments: the mitochondria and the cytosol. The first enzyme in the haem pathway, mitochondrial 8-aminolaevulinic acid synthase (8-ALA-S), forms 6-ALA from glycine and succinyl CoA and is a target for the regulation of haem biosynthesis. Feedback inhibition of 8-ALA-S occurs when intracellular haem is present in excess (Ade, 1990).The last step in the haem biosynthetic pathway, the insertion of iron into PPIX to form haem, is catalysed by the mitochondrial enzyme ferrochelatase (FC). In unperturbed systems, FC is regulated by the availability of iron and/or PPIX. Two of the metabolic events that occur between 8-ALA-S and FC are catalysed by the cytosolic enzymes 8-ALA dehydratase (8-ALA-D) and porphobilinogen deaminase (PBGD). The dehydratase enzyme catalyses the condensation of two 8-ALA molecules to form porphobilinogen (PBG). It is the first enzyme that will metabolize the administered 8-ALA. The next enzyme in haem biosynthesis, PBGD, forms the tetrapyrole ring from four porphobilinogen molecules (Abraham, 1991).By providing the 8-ALA-S product, 8-ALA, the initial feedback step has been circumvented and this approach has been exploited for use in photodynamic therapy (PDT) of cancer (Kennedy and Pottier, 1992;Grant et al, 1993; Cairnduff et al, 1994;Regula et al, 1995). Traditional PDT regimens consist of the systemic adminis- Accepted 13 June 1997 Correspondence to: R Hilf tration of a photosensitizer followed by an appropriate interval to allow for its maximal accumulation in ma...

Section: Discussionmentioning

confidence: 76%

A regulatory role for porphobilinogen deaminase (PBGD) in δ-aminolaevulinic acid (δ-ALA)-induced photosensitization?

Gibson

Cupriks²,

Havens³

et al. 1998

“…It is known that the activity of two enzymes, porphobilinogen deaminase and ferrochelatase, can be changed in malignant tissues (van Hillegersberg et al, 1992). This may result in a steeper rate of fluorescence increase in T and AS compared with NS after both topically and systemically administered ALA. That a disparity in enzymatic activity is an important factor in a different rate of fluorescence increase was supported by a previous study with a different animal model (van der Veen et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

“…For example, various malignant tissues have an increased porphobilinogen deaminase activity, which converts ALA into porphobilinogen, and a decreased ferrochelatase activity, which converts PpIX into haem (van Hillegersberg et al, 1992). This alteration in enzymatic activities may cause an increased fluorescence in tumour tissue compared with normal tissue after ALA administration (van der Veen et al, 1994;Bedwell et al, 1992).…”

mentioning

confidence: 99%

Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application, observed in skin and skin tumours of UVB-treated mice

Veen¹,

Bruijn²,

Berg³

et al. 1996

SummaryIn this study the kinetics and localisation of protoporphyrin IX (PpIX) fluorescence in skin and skin tumours were examined after topically (20% for 4 h) or systemically (200 mg kg-', i.p.) administered 5-aminolaevulinic acid (ALA). As a model we used hairless mice with skin lesions (actinic keratoses and squamous cell carcinoma), which were induced by daily UVB irradiation. The epidermis of the skin surrounding the tumours (T) was altered (AS); owing to the UVB irradiation, the epidermis was thicker and less elastic. Therefore, non-UVBirradiated mice were used to assess fluorescence of normal skin (NS). Light from a halogen lamp was used to excite at 500 + 20 nm and fluorescence was detected through a filter that passes light of 670 + 50 nm. Maximal fluorescence following i.p. ALA was observed 2 h post injection (p.i.) and was three times less than after topically applied ALA. Furthermore, after i.p. ALA a lower T selectively (T/NS) could be obtained than after topically applied ALA. Maximal fluorescence following topically applied ALA was achieved 6 h after the end of the 4 h application time. At that interval fluorescence of T was twice as high as directly after the application period. Furthermore, T selectivity (T/NS) after topical ALA at the interval of maximal fluorescence was higher than at the interval directly after application. With fluorescence cryomicroscopy localisation of fluorescence in the skin at the interval of maximal fluorescence was determined after both administration routes. For both cases fluorescence was mainly located in T, epidermis and hair follicles. Fluorescence in subcutis could only be observed at 2 h post i.p. ALA and at 6 h post topical ALA. No fluorescence could be observed in muscle. We conclude that, in this model and with these ALA doses, a higher fluorescence intensity and selectivity (T/NS) was achieved after topically applied ALA than after systemically administered ALA. These results make topically applied ALA more favourable for ALA-PDT of superficial skin tumours in this model. In general these results imply that by optimising the time after ALA application the efficacy and selectivity of topical ALA-PDT for skin tumours may be improved.

“…adminisered ALA. After both ALA doses the rate of fluorescence increase in TI was higher than in ST. It is known that various malignant tissues have a higher activity of porphobilinogen deaminase (PBGD) and a decreased activity of ferrochelatase (van Hillegersberg et al, 1992). Therefore, it is likely that the higher rate of fluorescence increase in TI may represent a higher capacity for conversion of ALA to porphyrin or PpIX to haem or a combination of both.…”

Section: Fluorescence Kinetic Studiesmentioning

confidence: 99%

In vivo fluorescence kinetics and photodynamic therapy using 5-aminolaevulinic acid-induced porphyrin: increased damage after multiple irradiations

Veen¹,

Leengoed²,

Star³

1994

111

The kinetics of fluorescence in tumour (TT) and subcutaneous tissue (ST) and the vascular effects of photodynamic therapy (PDT) were studied using protoporphyrin IX (PpIX), endogenously generated after i.v. administration of 100 and 200 mg kg-1 5-aminolaevulinic acid (ALA). The experimental model was a rat skinfold observation chamber containing a thin layer of ST in which a small syngeneic mammary tumour grows in a sheet-like fashion. Maximum TT and ST fluorescence following 200 mg kg-1 ALA was twice the value after 100 mg kg-1 ALA, but the initial increase with time was the same for the two doses in both TT and ST. The fluorescence increase in ST was slower and the maximum fluorescence was less and appeared later than in TT. Photodynamic therapy was applied using green argon laser light (514.5 nm, 100 J cm-2). Three groups received a single light treatment at different intervals after administration of 100 or 200 mg kg-1 ALA. In these groups no correlation was found between the fluorescence intensities and the vascular damage following PDT. A fourth group was treated twice and before the second light treatment some fluorescence had reappeared after photobleaching due to the first treatment. Only with the double light treatment was lasting TT necrosis achieved, and for the first time with any photosensitiser in this model this was accomplished without complete ST necrosis. Images Figure 4